Tropism modified cancer terminator virus (ad.5/3 ctv;ad.5/3-ctv-m7)

ABSTRACT

A tropism modified cancer terminator virus (Ad.5/3-CTV; Ad.5/3-CTV-M7) has been found to have infectivity that is Coxsackie Adenoviral Receptor (CAR) independent. The Ad.5/3-CTV (Ad.5/3-CTV-M7) may be used alone or in combination with other therapeutic agents such as agents that augment reactive oxygen (ROS) production, HDAC inhibitors, MCL-1 inhibitors and Bcl-2 inhibitors to treat a variety of cancers particularly including malignant glioma (GBM), renal cancer, prostate cancer, and colorectal cancer.

BACKGROUND OF THE INVENTION Field of the Invention

The invention generally relates to improved methods of adenoviral cancertherapy. In particular, the invention provides adenoviral vectors thatare Coxsackie Adenoviral Receptor (CAR) independent, cancer-specific intheir replication and produce a systemically active anti-cancercytokine.

Background

Adenoviral vectors have shown great promise with respect to the deliveryand expression of therapeutic agents to cells. However, to date, thelevel of infectivity, and hence the efficacy, of adenoviral vectors isdetermined by the presence, in the targeted cells, of CoxsackieAdenoviral Receptors (CARs), which mediate viral entry into the cell. Inthis regard, it is particularly unfortunate that many types of cancercells express low or no appreciable levels of CARs. This feature ofcancer cells severely curtails the otherwise promising use of adenoviralvectors as anticancer agents. Additionally, an ability to selectivelyreplicate in cancer cells and also produce a therapeutically active andsafe secreted cytokine will limit non-specific toxicity and allow fortreatment of both primary infected tumors and distant infected ornon-infected metastases.

SUMMARY

The invention provides a novel chimeric tropism modified adenoviralvector, Ad.5/3-CTV (Ad. 5/3-CTV-M7) that displays profound anti-canceractivity in human cancer cells. Significantly, this chimera infectscancer cells in a CAR-independent manner, i.e., the anticancer agentsencoded by this adenoviral construct are expressed in both high and lowCAR cancer cells. As demonstrated herein, Ad.5/3-CTV (Ad. 5/3-CTV-M7)displays enhanced anti-tumor activity in vitro and in vivo in xenografttumors in nude mice in low CAR prostate cancer, glioma (GBM), renalcancer and colorectal cancer.

Ad.5/3-CTV (Ad.5/3-CTV-M7) is a new cancer terminator virus, which maybe used to treat a wide variety of cancers. Ad. 5/3-CTV (Ad. 5/3-CTV-M7)exhibits enhanced infectivity compared with other cancer terminatorviruses, e.g., viruses where adenoviral replication is controlled by thecancer-selective Progression Elevated Gene-3 (PEG-3) promoter and whichsimultaneously express an anticancer cytokine, such as melanomadifferentiation associated gene-7/Interleukin-24 (mda-7/IL-24) from theE3 region of the adenovirus. Ad.5/3-CTV (Ad. 5/3-CTV-M7) may beadministered by a variety of routes particularly including systemicadministration, alone or in combination with one more therapeutic agents(e.g., additional anticancer agents), together with a carrier (e.g.,aqueous fluid). Very good in vivo delivery results are achieved whendelivering Ad.5/3-CTV (Ad. 5/3-CTV-M7) associated with perfluorocarbonmicrobubbles as a carrier, where, after arrival at or near the cancertissue and its associated tumor vasculature, the microbubbles are burstby exposure to ultrasound (This approach is called ultrasound-targetedmicrobubble-destruction (UTMD)). Particularly good results may beachieved in specific cancers when the mda-7/IL-24 therapeutic geneproduct from the Ad.5/3-CTV (Ad. 5/3-CTV-M7) is combined with agentsthat augment reactive oxygen species (ROS) production, such as limoneneand perillyl alcohol. For example, these agents which augment ROSproduction enhance the therapeutic index of mda-7/IL-24 in pancreaticcancer cells. Combining mda-7/IL-24 produced from Ad.5/3-CTV(Ad.5/3-CTV-M7) with HDAC inhibitors such as SAHA or sodium valproaterepresent another way of enhancing therapeutic activity of mda-7/IL-24.The use of sabutoclax (an exemplary MCL-1 inhibitor) with the Ad.5/3-CTV (Ad.5/3-CTV-M7) enhances its activity against prostate and othercancers. Obatoclax (an exemplary Bcl-2 and Bcl-xL inhibitor) shouldconfer increased lethality to GBM cells when combined with Ad.5/3-CTV(Ad.5/3-CTV-M7). In addition, enhanced survival is achieved whenAd.5/3-CTV (Ad.5/3-CTV-M7) is used in combination with vorinostat(Zolinda) compared to either Ad.5/3-CTV (Ad.5/3-CTV-M7) or vorinostatalone.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Generation of a tropism-modified cancer terminator virus(Ad.5/3-CTV; Ad.5/3-CTV-M7). Schematic representation outlining theconstruction of a tropism modified cancer terminator virus for deliveryof mda-7/IL-24. The detailed procedures are described in Material andMethods of Example 1.

FIG. 2A-D. Ad.5/3-CTV (Ad.5/3-CTV-M7) enhances mda-7/IL-24 expressionand inhibition of cell viability in low CAR prostate cancer cells. A andC) DU-145 and PC-3 cells were infected with the indicated vp/cell ofAd.5-vec, Ad.5-PEG-E1A, Ad.5-CTV (Ad.5-CTV-M7), Ad.5/3-vec,Ad.5/3-PEG-E1A, and Ad.5/3-CTV (Ad.5/3-CTV-M7) for 48 hr and totalproteins were isolated. The expression of MDA-7/IL-24 (23.8-kDa protein,with 35- to 40-kDa glycosylated species detected on the gel), E1A andEF-1α (as a loading control) proteins were analyzed by Western blotanalyses. B and D) Cell viability using the MTT assay was quantifiedafter 3 days and 6 days with the indicated doses of vp/cell of Ad.5S-CTV(Ad.5-CTV-M7), Ad.5/3-CTV (Ad.5/3-CTV-M7) and their respective controls,C and D. Results are the mean±S.D. (n=3).

FIG. 3A-C. Ad.5/3-CTV (Ad.5/3-CTV-M7), but not Ad.5-CTV (Ad.5-CTV-M7),induce ER stress and apoptosis, and overcome therapy resistance inPC-3-Bcl-2 tumor cells. a) Changes in BiP/GRP78, GRP94 and activation ofPARP were detected by Western blot analysis after 2 days of treatment ofPC-3 cells with the indicated Ads. b+c) Cell proliferation and viabilityusing the MTT assay was quantified after 6 days with the indicated dosesof vp/cell of Ad.5-CTV (Ad.5-CTV-M7), Ad.5/3-CTV (Ad.5/3-CTV-M7) andtheir respective controls. Results are the mean±S.D. (n=3). *, P<0.05with the Ad.5-vec 10000 vp/cell infected group.

FIG. 4A-E. Ad.5/3-CTV (Ad.5/3-CTV-M7) eradicates primary and inhibitsdistant PC-3-Bcl-2 xenografts in nude mice. Tumor xenografts withPC-3-Bcl-2 cells were established in athymic nude mice in both right andleft flanks; and only tumors on the left side were injected with theindicated Ads over a 4-week period (total of nine injections).Measurements of PC-3-Bcl-2 xenograft tumor volumes on A) left and B)right flanks; points, average (with a minimum of five mice in eachgroup); bars, ±S.D. Inset contains a photograph of the animals of eachrepresentative group. C) Photograph of the PC-3-Bcl-2 xenograft tumor atthe end of the study. D) Measurement of tumor weight at the end of thestudy; columns, mean (with at least five mice in each group); bars,±S.D. E) Western blot analysis of protein extracts from representativePC-3-Bcl-2 tumor samples treated with Ad.5-vec, Ad.5/3-vec,Ad.5-PEG-E1A, Ad.5/3-PEG-E1A, Ad.5-CTV (Ad.5-CTV-M7), Ad.5/3-CTV (Ad.5/3-CTV-M7). The immunoblot was reacted with anti-MDA-7/IL-24.

FIG. 5A-C: Combination treatment of Ad.5/3-CTV (Ad.5/3-CTV-M7) andBI-97C1 (Sabutoclax) potentiates inhibition of prostate tumor growth invivo in immune competent animals. A): The prostatic region of 22week-old male Hi-Myc mice were sonoporated for 10 min followingtail-vein injection of the indicated complement-treated microbubble/Adcomplexes and treated as described in the materials and methods sectionfor 4 weeks (total 8 injections of indicated viruses). BI-97C1(Sabutoclax) was administered intraperitoneally (i.p.) in each group at3 mg/kg 3× a week throughout the study. At the end of the experiment themice were sacrificed and the prostates were collected. Theparaffin-embedded sections were obtained from the prostate andimmunohistochemistry was performed to measure systemic transgenedelivery by staining with anti-MDA-7/IL-24. TUNEL assay and Ki-67staining detected apoptosis and cell proliferation in the prostatesections. Nuclei were visualized with DAPI. Wild type mice of the samestrain that do not develop prostate cancer served as a control for theseexperiments. B) Quantification of microvessel density in prostatesection per field followed by Ki-67 staining and C) Quantification ofTUNEL positive signals in the prostate section (*P<0.05 between theindicated groups). Data represent mean±S.D. (n=3).

FIG. 6A-D. PEG-Prom is selectively more active in pancreatic cancercells than normal LT-2 cell lines. A) Pancreatic cells were treated withAd.PEG-GFP at 5000 vp/cell and then incubated for additional 48 h forits expression. (A) Images were taken with a 20× magnification lensunder a fluorescent microscope. (B) Cells treated with Ad.vec (solidfilled black line) or Ad.PEG-GFP (solid red line) were collected andGFP-expression was measured using a flow-cytometer. (C) The relativeexpressed GFP or fluorescence values (RFU; Ad.5.PEG-GFP/Ad.5.CMV-GFP)were quantified using FacsDIVA software. (D) Relative Luminescence units(RLU) of pGL3.PEG-luc/pGL3.CMV-luc after transfecting the cells withpGL3.PEG-luc and pGL3.CMV-luc. ***, p<0.001 versus RFU of infected orRLU of transfected LT-2. The data represents mean±S.E of three differentexperiments.

FIGS. 7A and B. Ad.5-PEG-E1A-mda-7 (Ad.5-CTV-M7) or Ad.5/3-PEG-E1A-mda-7(Ad.5/3-CTV-M7) and POH synergistically inhibit in vitro growth ofpancreatic cancers. Both wild type and mutant K-Ras pancreatic cancercells, i.e., BxPC-3 (A) and MIA PaCa-2 (B), respectively, were infectedwith Ad.5-mda-7, Ad.5/3-mda-7, Ad.5-CTV-M7 or Ad.5/3-CTV-M7 followed bytreatment with or without POH and MTT assays were performed after 3days. Points; mean±S.E of three different experiments each performed inquadruplicate.

FIG. 8. Enhanced mda-7/IL-24-induced apoptosis in pancreatic cell linesby Ad.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV-M7) in combination with POH. MIAPaCa-2 and BxPC-3 cells were treated as indicated for 48 h. Cells werecollected, lysed, and proteins were separated by SDS-PAGE and analyzedby Western blotting. EF-1α was used as loading control.

FIG. 9A-F. ROS generated by POH induces increased expression ofMDA-7/IL-24 in pancreatic cancer cell lines culminating in enhancedapoptosis. (A) MIA PaCa-2 and (B) BxPC-3 cell lines were infected withthe indicated Ads and/or POH, and secreted MDA-7/IL-24 was quantifiedusing human MDA-7/IL-24 ELISA kit, and mda-7/IL-24 mRNA from (C) MIAPaCa-2 and (D) BxPC-3 was quantified using qRT-PCR 48 h post infection.Apoptosis in (E) MIA PaCa-2 and (F) BxPC-3 cells was measured byflow-cytometer after staining the cells with AnnexinV/PI. *(p<0.05),**(p<0.01), and ***(p<0.001) indicates the increasing level ofsignificance after performing un-paired t-test between Ads treated groupvs. Ads treated group plus POH as indicated in A, B, E and F. *(p<0.05),**(p<0.01), and ***(p<0.001) indicates the increasing level ofsignificance after performing an un-paired t-test of different Adstreated group with respect to Ad.5-mda-7 treated group as shown in C andD.

FIG. 10A-G. Ad.5/3-CTV-M7 sensitizes therapy-resistant pancreatic cellsto cell killing by inducing ER-stress. MIA PaCa-2 clones stablyexpressing (A) Bcl-2 and (B) Bcl-xL were developed and quantificationwas done by qPCR. *(p<0.05), **(p<0.01), and ***(p<0.001) indicates theincreasing level of significance after performing un-paired t-testbetween control vs. different stable clones. MIA PaCa-2 clones stablyexpressing (C) Bcl-2 and (D) Bcl-xL stable were treated withAd.5/3-mda-7, Ad.5/3-mda-7+POH, Ad.5/3-CTV-M7, Ad.5/3-CTV-M7+POH oruntreated Ad.5/3-vec (control). *, p<0.05 versus Ads treated cl-1(control clone for Bcl-2 overexpression) (C) or cl-6 (control clone forBcl-xL overexpression) (D) cells. (E) Overexpression of Bcl-xL inhibitsThapsigargin (Thap)-induced ER stress-mediated apoptosis as compared tocontrol pcDNA 3.1 MIA PaCa-2 cell cl (con). (F) MIA PaCa-2/Bcl-xL cl-3(stable clone expressing the maximum level of Bcl-xL) cells were treatedwith BiP/GRP-78 shRNA (sh-BiP) followed by treatment with Ad and/or POH.Apoptosis was measured 48 h post-infection by flow-cytometer afterstaining the cells with AnnexinV/PI and (G) Western blotting of MIAPaCa-2/Bcl-xL (cl-3) cells untreated or treated with POH and infectedwith the indicated Ads was performed after probing with the indicatedantibodies.

FIG. 11A-E. Ad.5/3-CTV-M7 in combination with POH completely eradicatesprimary and distant tumors in nude mice bearing human pancreatic tumorxenografts. Tumor xenografts generated from MIA-PaCa-2 cells stablyexpressing a luciferase gene (MIA PaCa-2-luc) were established in boththe left and right flanks of nude mice. Tumors on the left flank weresonoporated for 10 min following tail-vein injection of the indicatedcomplement treated Ad/MB complex, and treated as described in Materialsand Methods section for 3 weeks starting from day 14 (1 injection/week).POH was administered i.p. daily for 4 weeks from day 1 of implantationof cells in nude mice. (A) Bioluminescence imaging (BLI) was performedusing Xenogen In Vivo Imaging System (IVIS). BLI was performed everyweek and luminescence was quantified and tumor growth response curve ofthe left flank (B) and right flank or distant tumor (C) was plotted. (D)Secreted MDA-7/IL-24 in blood serum was measured using an MDA-7/IL-24ELISA kit. (E) There was significant increase in ‘bystander’ effect ofMDA-7/IL-24 in inhibiting in vivo tumor growth as measured by BLI.Statistical analyses were performed using un-paired t-test betweendifferent groups. *, **, and *** indicates the level of significancewith p<0.05, p<0.01 and p<0.001 respectively.

FIG. 12A-E. In vivo antitumor effect of Ad.5/3-CTV-M7 plus POH in nudemice bearing therapy resistant MIA PaCa-2/Bcl-xL xenografts. Tumorxenografts with MIA-PaCa-2 cells stably expressing a Bcl-xL gene (MIAPaCa-2/Bcl-xL) were established in both the left and right flanks.Tumors on the left flank were sonoporated for 10 min following tail-veininjection of indicated complement treated Ad/microbubble complex, andtreated as described in Materials and Methods section for 3 wks startingfrom day 14 (1 injection/week). POH was administered i.p. injectiondaily for 4 weeks from day 1 of implantation of cells in nude mice.Tumor volume of the left flank (A) and right flank (B) was measuredusing digital calipers, and the graph was plotted over time. Tumor massand weight were measured for both left (C) and right flank (D) tumorsafter the mice were sacrificed at the end of experiment. (E) Apoptosiswas measured by TUNEL staining of fixed cells. Statistical analyses wereperformed using un-paired t-test between different groups. *(p>0.05),**(p>0.01), and ***(p>0.001) versus Ad.5/3-treated group.

FIGS. 13A-D. Cancer-specific oncolytic effects of CTV-M7 in pancreaticcancer cells. Pancreatic cancer cells were treated with non-replicatingAd.5-mda-7 or Ad.5/3-mda-7 as well as conditional replication-competentAds (CRCA) i.e., Ad.5-CTV-M7 and Ad.5/3-CTV-M7 and MTT assays wereperformed after 3 days. IC₅₀ was calculated using GraphPad Prism 5.0.Points; mean±S.E. of three different experiments each performed inquadruplicate.

FIG. 14. Temporal kinetics of reactive oxygen species (ROS) formationinduced by POH. MIA PaCa-2 and BxPC-3 cells were stained withcarboxy-H2DCF-DA in PBS for 30 min followed by treatment of POH, andfluorescence was measured using a Flurometer with a green filter at theindicated time points. The readings noted were the difference betweenPOH-treated group compared to untreated control.

FIGS. 15A-D. Combination index or Interaction index of pancreatic cancercells treated with CTV-M7 plus POH. BxPC-3 and MIA PaCa-2 cells weretreated with increasing doses (vp/cell) of Ad.5-CTV-M7 or Ad.5/3-CTV-M7(10-1000) in combination with increasing doses of POH (10-1000 μM) andMTT results were plotted to calculate the combination index (CI).

FIG. 16. Bioluminescence imaging (BLI) indicates a positive correlationbetween bioluminescence and cell number. MIA PaCa-2-luc cells wereseeded in 24-well plate, and luminescence was measured after 12 h afteradding D-luciferin. Image acquisition was performed using a Xenogen InVivo Imaging System (IVIS) and the images were quantified using LivingImage 4.3.1 software.

FIG. 17. Ad.5/3-CTV-M7 eradicates primary and secondary tumors in vivoin a human pancreatic cancer xenograft model. Tumor xenografts of MIAPaCa-2 cells stably expressing a luciferase gene (MIA PaCa-2-luc) wereestablished in both the left and right flanks of athymic nude mice.Tumors on the left flank were sonoporated for 10 min following tail-veininjection of the indicated complement treated Ad/MB complexes, andtreated as described in Materials and Methods section for 3 weeksstarting from day 14 (1 injection/week). POH was administered i.p. dailyfor 4 weeks from day 1 of impanation of cells in nude mice.Bioluminescence imaging (BLI) was performed using a Xenogen In VivoImaging System (IVIS) after i.p. administration of D-luciferin. BLI wasperformed at the indicated days.

Figures for Example 3

FIG. 18A-C. SBHA and Na Valproate enhance Ad.5-mda-7 toxicity inmultiple primary human GBM isolates but not in primary human astrocytes.(A) GBM6 cells, (B) GBM12 cells and (C) primary human astrocytes wereinfected with empty vector or recombinant serotype 5 adenovirus toexpress MDA-7/IL-24; at a multiplicity of infection (moi) of 10. Afterinfection (24 h) cells were treated with vehicle control or withpharmacologically achievable concentrations of HDACIs, Na Valproate (0,0.3, 0.5, 1.0 mM) or the vorinostat analogue SBHA (0, 0.3, 1.0, 3.0, 5.0□DM). Cells were isolated 48 h later and viability determined by trypanblue exclusion (n=3, +/−SEM) (*p<0.05 greater than corresponding valuein Ad.5-cmv infected cells).

FIG. 19A-D. Induction of ER stress and autophagy plays a role in theinteraction between MDA-7/IL-24 and HDACIs. Panel A. GBM6 cells weretransfected in quadruplicate with a plasmid to express an LC3 (ATG8)-GFPfusion protein and in parallel transfected with scrambled siRNA (siSCR)or an siRNA to knock down Beclin1 expression. Twenty-four h aftertransfection cells were treated with GST or GST-MDA-7 (20 nM) and/orSBHA (3 μM). Cells (a representative of 40 per well per time point) wereexamined 6 h, 12 h and 24 h after treatment using an Axiovert microscope(×40) for the formation of punctuate vesicles containing LC3-GFP. Dataare plotted as the number of LC3-GFP vesicles per cell (n=2, +/−SEM)(*p<0.05 greater than GST+VEH; **p<0.05 greater than GST-MDA-7+VEH).Panel B. GBM6 cells were transfected with scrambled siRNA (siSCR) or ansiRNA to knock down Beclin1 expression. Twenty-four h after transfectioncells were treated with GST or GST-MDA-7 (20 nM) and/or SBHA (3 μM).Cells were isolated 48 h later and viability determined by trypan blueexclusion (n=3, +/−SEM) (# p<0.05 less than corresponding value in siSCRcells). Panel C. GBM6 cells were transfected with an empty vectorcontrol plasmid or a plasmid to express dominant negative PERK.Twenty-four h after transfection cells were treated with GST-MDA-7 (20nM), SBHA (3 M) or the agents combined. Cells were isolated 6 h afterexposure and the phosphorylation of PERK and eIF2α determined(representative n=2). Panel D. GBM6 cells were transfected inquadruplicate with an LC3 (ATG8)-GFP fusion protein and in paralleltransfected with an empty vector control plasmid or a plasmid to expressdominant negative PERK. Twenty-four h after transfection cells weretreated with GST or GST-MDA-7 (20 nM) and/or SBHA (3 μM). Cells (arepresentative of 40 per well per time point) were examined 24 h aftertreatment using an Axiovert microscope (×40) for the formation ofpunctuate vesicles containing LC3-GFP. Data are plotted as the number ofLC3-GFP vesicles per cell (n=3, +/−SEM) (# p<0.05 less thancorresponding value in CMV cells).

FIG. 20. GBM6 cells were transfected with an empty vector controlplasmid or a plasmid to express dominant negative PERK. Twenty-four hafter transfection cells were treated with GST-MDA-7 (20 nM), SBHA (3μM) or the agents combined. Cells were isolated 48 h later and viabilitydetermined by trypan blue exclusion (n=3, +/−SEM) (#p<0.05 less thancorresponding value in CMV cells).

FIG. 21A-B. De novo ceramide generation plays an essential role in theinteraction between MDA-7/IL-24 and HDACIs. Panel A. GBM6 cells weretransfected in quadruplicate with a plasmid to express an LC3 (ATG8)-GFPfusion protein and in parallel transfected with scrambled siRNA (siSCR)or an siRNA to knock down ceramide synthase 6 (LASS6) expression. Cellswere infected with empty vector or recombinant serotype 5 adenovirus toexpress MDA-7/IL-24; at a multiplicity of infection (moi) of 10. Afterinfection (24 h) cells were treated with vehicle control or with thevorinostat analogue SBHA (3.0 μM). Cells (a representative of 40 perwell per time point) were examined 24 h after treatment using anAxiovert microscope (×40) for the formation of punctuate vesiclescontaining LC3-GFP. Data are plotted as the number of LC3-GFP vesiclesper cell (n=3, +/−SEM) (# p<0.05 less than corresponding value in siSCRcells). Panel B. GBM6 and GBM14 cells were transfected with scrambledsiRNA (siSCR) or an siRNA to knock down ceramide synthase 6 (LASS6)expression. Twenty-four h later cells were infected with empty vector orrecombinant serotype 5 adenovirus to express MDA-7/IL-24; at amultiplicity of infection (moi) of 10. After infection (24 h) cells weretreated with vehicle control or with the vorinostat analogue SBHA (3.0μM). Cells were isolated 48 h later and viability determined by trypanblue exclusion (n=3, +/−SEM) (# p<0.05 less than corresponding value insiSCR cells).

FIG. 22A-C. SBHA intensifies and prolongs ROS and Ca²⁺ generation causedby MDA-7/IL-24. Panel A. GBM6 cells were infected with empty vector orrecombinant serotype 5 adenovirus to express MDA-7/IL-24; at amultiplicity of infection (moi) of 10. Twenty-four h after infectioncells were treated with vehicle (DMSO) or SBHA (3 μM). Cells were loadedwith DCFH and the levels of ROS under each condition determined 6 h, 12h and 24 h after SBHA treatment (n=3, +/−SEM) (*p<0.05 greater thanAd.5-mda-7+VEH). Panel B. GBM6 cells were infected with empty vector orrecombinant serotype 5 adenovirus to express MDA-7/IL-24; at amultiplicity of infection (moi) of 10. Twenty-four h after infectioncells were treated with vehicle (DMSO) or SBHA (3 μM). Cells were loadedwith Fura-2 and the levels of free Ca²⁺ under each condition determined6 h, 12 h and 24 h after SBHA treatment (n=3, +/−SEM) (*p<0.05 greaterthan Ad.5-mda-7+VEH). Panel C. GBM6 cells were transfected with an emptyvector plasmid, a plasmid to express thioredoxin (TRX) or a plasmid toexpress calbindin and in parallel infected with empty vector orrecombinant serotype 5 adenovirus to express MDA-7/IL-24; at amultiplicity of infection (moi) of 10. Twenty-four h after infectioncells were treated with vehicle (DMSO) or SBHA (3 μM). Cells wereisolated 48 h later and viability determined by trypan blue exclusion(n=3, +/−SEM). (# p<0.05 less than corresponding value in CMVtransfected cells).

FIGS. 23A and B. SBHA enhances MDA-7/IL-24 toxicity through theextrinsic pathway. Panel A. GBM6 cells were infected with empty vectoror recombinant serotype 5 adenovirus to express c-FLIP-s; CRM A; BCL-XL;or dominant negative caspase 9. Twenty-four h after infection cells weretreated with GST or GST-MDA-7 (20 nM) and/or SBHA (3 μM). Cells wereisolated 48 h later and viability determined by trypan blue exclusion(n=3, +/−SEM). (# p<0.05 less than corresponding value in CMV infectedcells; ¶ p<0.05 less than corresponding values in CMV, c-FLIP-s and CRMA infected cells). Panel B. GBM6 cells were infected with empty vectoror recombinant serotype 5 adenovirus to express MDA-7/IL-24; at amultiplicity of infection (moi) of 10. Twenty-four h after infectioncells were treated with increasing concentrations of obatoclax(GX15-070, 0-200 nM) or HA14-1 (0-10 μM). Cells were isolated 24 h laterand viability determined by trypan blue exclusion (n=3, +/−SEM) (*p<0.05greater than corresponding Ad.5-cmv values).

FIG. 24. SAHA enhances MDA-7/IL-24 toxicity in vivo. GBM6 cells(0.5×10⁶) were implanted into the brains of athymic mice. Seven dayslater tumors were infused with 1×10⁸ pfu of either Ad.5/3-cmv orAd.5/3-mda-7. Twenty-four h after virus infusion animals were treatedwith vehicle diluent (cremophore, PO) or vorinostat (SAHA, 25 mg/kg QD,PO) for 5 days. Animal survival was monitored on a daily basis (n=2, 8animals total).

FIG. 25A-E. Ad.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV, Ad.5/3-CTV-M7) prolongsanimal survival in a dose-dependent fashion and does so to a greaterextent than Ad.5/3-mda-7. Panel A. GBM6-luciferase cells (0.5×10⁶) wereimplanted into the brains of athymic mice. Seven days later tumors wereinfused with 1×10⁸ pfu of either: Ad.5/3-cmv; Ad.5/3-mda-7;Ad.5/3-PEG-E1A; or Ad.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV; Ad.5/3-CTV-M7). Onthe days indicated in the graph animals were injected with luciferin(150 mg/kg) and were imaged 15 min later following placement into anIVIS Xenogen imager. The fold-increase in luciferase intensity for themean of each animal group was plotted (n=2, 6 animals total+/−SEM) (#p<0.05 less than Ad.5/3-cmv; Ad.5/3-mda-7; or Ad.5/3-PEG-E1A (Ad.5/3-CTV; Ad.5/3-CTV-M7). The Fold change in luciferase activity at day12 is shown numerically. Panel B. Brains from animals at day 12 (PanelA) were removed, were fixed in OCT compound (Tissue Tek); cryostatsectioned (Leica) as 12 μm sections. Sections from tumor tissue andnormal brain were stained for apoptosis (TUNEL) and for expression ofthe viral E1A protein. Panel C. GBM6 cells (0.5×10⁶) were implanted intothe brains of athymic mice. Seven days later tumors were infused with:Ad.5/3-cmv (1×10⁹ pfu); Ad.5/3-mda-7 (1×10⁹ pfu); Ad.5/3-PEG-E1A (1×10⁸;3×10⁸; 1×10⁹ pfu); and Ad.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV Ad.5/3-CTV-M7)(1×10⁸; 3×10⁸; 1×10⁹ pfu). Animal survival was monitored on a dailybasis (n=2, 6 animals total) ((a) p<0.04 greater survival thanAd.5/3-cmv; (b) p<0.0008 greater survival than Ad.5/3-cmv; (c) p<0.04greater survival than Ad.5/3-mda-7; (d) p<0.004 greater survival thanAd.5/3-mda-7; (e) p<0.0008 greater survival than Ad.5/3-PEG-E1A-mda-7(Ad.5/3-CTV Ad.5/3-CTV-M7) at a dose of 3×10⁸ pfu). Panel D. Syrianhamster brains were infused with PBS; Ad.5/3-cmv-E1A (2×10⁹ pfu); orAd.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV; Ad.5/3-CTV-M7) (2×10⁹ pfu).Seventy-two h after infusion animal brains, livers and kidneys wereisolated and fixed. Sections (12 μm) were taken and stained forapoptosis (TUNEL), the levels of viral E1A protein, and the levels ofMDA-7/IL-24 protein. Panel E. Syrian hamster brains were infused withPBS; Ad.5/3-cmv-E1A (2×10⁹ pfu); or Ad.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV;Ad.5/3-CTV-M7) (2×10⁹ pfu). Seventy-two h and 1 week after infusionanimals were sacrificed and their neck lymph nodes dissected.

FIGS. 26A-G Full Length Sequences for (1) PEG-3-E1, (2) CMVp-mda-7 and(3) Ad.5/3 fiber.

DETAILED DESCRIPTION

The invention provides a novel chimeric tropism modified adenoviralvector, Ad.5/3-CTV (also referred to as Ad.5/3-CTV-M7, a tropismmodified CTV (containing mda-7) in the Ad.5/3 background) with profoundanti-cancer activity in both low and high CAR expression human cancercells.

Ad.5/3-CTV (Ad. 5/3-CTV-M7) is based in part on a previously constructedconditionally replication competent adenovirus (CRACA), “CancerTerminator Virus” (CTV). In CTV, adenoviral replication is controlled bythe cancer-selective Progression Elevated Gene-3 (PEG-3) promoter andthis construct simultaneously expresses the anticancer agent melanomadifferentiation associated gene-7 also called interleukin-24(mda-7/IL-24) from the E3 region of the adenovirus (Ad. 5/3-CTV-M7).However, CTV was generated on a serotype 5-background (Ad.5-CTV) so thatinfectivity depends on the presence of Coxsackie-Adenovirus Receptors(CARs) in targeted cells. CARs are frequently reduced in many tumortypes, including malignant gliomas (GBM), renal cancers, prostatecancer, colorectal cancer and many others, thereby limiting effectivetherapy.

To develop methods for improving infectivity of human cancers,engineered variant adenoviruses (Ads) from different species wereconstructed and evaluated. These studies produced the unanticipatedfinding that serotype chimerism created by replacing the Ad.5 fiber knobwith the Ad.3 fiber knob resulted in viruses with superior infectivityof diverse human cancer cell types. Based on this observation, a noveladenoviral construct has now been created using the Ad.5/3 chimerism incombination with CTV (Ad.5/3-CTV Ad.5/3-CTV-M7). This chimeraadvantageously infects cancer cells in a CAR-independent manner. Asdemonstrated herein, Ad.5/3-CTV (Ad.5/3-CTV-M7) displays enhancedanti-tumor activity in vitro and in vivo in xenograft tumors in nudemice, even in low CAR prostate, GBM, renal and colorectal cancers.Additionally, Ad.5/3-CTV (Ad.5/3-CTV-M7) shows significant anti-tumoractivity in immune competent Hi-Myc transgenic mice, which developprostate cancer, e.g., when delivered by ultrasound-targeted microbubbledestruction (UTMD) approach.

The invention thus provides the genetically engineered adenoviral vectorAd.5/3-CTV (Ad.5/3-CTV-M7) as well as compositions containing the vectorand methods of using the same to kill cancer cells and treat cancer,especially cancers which express little or no CARs. Ad.5/3-CTV(Ad.5/3-CTV-M7) thus has utility in addressing a wide array of differenttypes of cancers, and it may be used alone or in combination with othertherapeutic agents.

Specific examples of cancers that may be treated with Ad.5/3-CTV (Ad.5/3-CTV-M7) alone or in combination with other therapeutic agentsinclude but are not limited to solid tumors, blood born tumors such asleukemia, acute or chronic lymphoblastic leukemia, breast cancer,chordoma, craniopharyngioma, endometrial cancer, ependymoma, Ewing'stumor, gastric cancer, germinoma, glioma, glioblastoma,hemangioblastoma, hemangioperycatioma, Hodgkins lymphoma,medulloblastoma, leukaemia, mesothelioma, neuroblastoma, non-Hodgkinslymphoma, pinealoma, retinoblastoma, sarcoma (including angiosarcoma,osteosarcoma and chondrosarcoma), bladder carcinoma, brain tumor, breastcarcinoma, bronchogenic carcinoma, carcinoma of the kidney, cervicalcarcinoma, choriocarcinoma, cystadenocarcinome, embryonal carcinoma,epithelial carcinoma, esophageal carcinoma, cervical carcinoma, coloncarcinoma, colorectal carcinoma, endometrial carcinoma, gallbladdercarcinoma, gastric carcinoma, head and neck carcinoma, liver carcinoma,lung carcinoma, medullary carcinoma, non-small cell bronchogenic/lungcarcinoma, lung cancer, ovarian carcinoma, pancreas carcinoma, papillarycarcinoma, papillary adenocarcinoma, prostate carcinoma, small intestinecarcinoma, rectal carcinoma, renal cell carcinoma, skin carcinoma,squamous cell carcinoma, sebaceous gland carcinoma, testicularcarcinoma, osteosarcoma, ovary cancer, uterine carcinoma, CAR prostatecancer, glioma (GBM), renal cancer, and colorectal cancer.

Specific examples of therapeutic agents, which may be used incombination with Ad.5/3-CTV (Ad.5/3-CTV-M7) include but are not limitedto agents that augment reactive oxygen (ROS) production (ROS inducers,natural products and other agents) (e.g., limonene, perillyl alcohol,arsenic trioxide, resveratrol, cyaniding-3-rutinoside, diallyl disulfide(DADS), and methyl jasmonate), HDAC inhibitors (e.g., SAHA, Vorinostat,Rocllinostat (ACY-1215) Panobinostat (LBH589), Entinostat (MS-275),Romidepsin (FK228, Depsipeptide), Trichostatin A (TSA), Mocetinostat9MGCD0103) RGFP966, Bellinostat (PXD101), Scriptald, PCL-24781(Abexinostat), LAQ824 (Dacinostat), JNJ26481585, valproic acid sodiumsalt (sodium valproate), CUDC-101, Drosinostate, MC1568, Pracinostate(SB939), Givinostat (ITF2357) AR-42, Tubostatin A HCl, PCI-34051,CUDC-907, M344, CI994 (Tacedinaline), Tubostatin A, sodiumphenylbutyrate, and Resminostat), MCL-1 inhibitors (e.g., sabutoclax,BI-97D6, Gambogic acid,4-((E)-((Z)-2-(cyclohexylimino)-4-methylthiazol-3(2H)-ylimino)methyl)benzene-1,2, 3-triol), and Bcl-2/BCL-xL inhibitors (e.g., ABT-737 (BCL-xLinhibitor), oblimersen sodium, AT-101, ABT-263, GX15-070, HA14-1, andObatoclax).

As discussed in the Examples, and particularly with reference to Example1 and FIG. 1, Ad.5/3-CTV (Ad.5/3-CTV-M7) may be constructed by 1)Homologous recombination of pAd.5/3 genomic plasmid with pShuttlE3plasmid containing the mda-7/IL-24 expression cassette and kanamycinselection results in the pAd.5/3-E3-mda-7 genome. 2) pAd.5/3-E3-mda-7was cut with Swa I to excise the kanamycin resistance gene. 3) Theresultant pAd.5/3-E3-mda-7 plasmid was recombined with pShuttlE1 plasmidcontaining E1A and E1B genes under control of the PEG-3 promoterresulting in Ad.5/3-PEG-E1-mda-7 (Ad.5/3-CTV, Ad.5/3-CTV-M7) genomicplasmid. This plasmid was digested with Pac I to release viral ITRs andtransfected in A549 cells to rescue the CRCA, Ad.5/3-CTV.

The nucleic acid sequences of (1) PEG-3-E1, (2) CMVp-mda-7 and (3) theAd.5/3 fiber of Ad.5/3-CTV are presented in FIG. 26 and are set forth asSEQ ID NO: 1 (PEG-3-E1), SEQ ID NO: 2 (CMVp-mda-7) and SEQ ID NO: 3(Ad.5/3 fiber).

The invention also provides methods of treating cancer by delivering toa patient in need thereof, a therapeutically effective amount of theAd.5/3-CTV (Ad.5/3-CTV-M7) vector, alone or in combination with one ormore therapeutic agent(s), to the patient. The subject to whom theadenoviral vector is administered is usually a mammal, and is generallya human, although this need not always be the case as veterinaryapplications of this technology are also contemplated. If the Ad.5/3-CTV(Ad.5/3-CTV-M7) is combined with one or more additional therapeuticagents for provisioning to a subject in need thereof, these additionaltherapeutic agents can be provided simultaneously with the Ad.5/3-CTV(Ad. 5/3-CTV-M7) or before or after the provisioning of the Ad. 5/3-CTV(Ad. 5/3-CTV-M7). Further, the one or more additional therapeutic agentsmay be provided by the same route of administration as the Ad.5/3-CTV(Ad.5/3-CTV-M7) or by one or more different routes.

Any route of administration can be used to deliver the adenoviral vectorto the mammal. Indeed, although more than one route can be used toadminister the adenoviral vector, a particular route can provide a moreimmediate and more effective reaction than another route. The adenoviralvector may be administered intravenously or via intratumoral injection.A dose of adenoviral vector also can be applied or instilled into bodycavities, absorbed through the skin (e.g., via a transdermal patch),inhaled, ingested, topically applied to tissue, or administeredparenterally via, for instance, intravenous, peritoneal, intramuscular,or intraarterial administration. In one aspect, the adenoviral vector isadministered via ultrasound microbubble technology, as described in USpatent application 2012/0195935 (Fisher et al), which is hereinincorporated by reference.

The adenoviral vector can be administered in or on a device that allowscontrolled or sustained release, such as a sponge, biocompatiblemeshwork, mechanical reservoir, or mechanical implant. Implants (see,e.g., U.S. Pat. No. 5,443,505), devices (see, e.g., U.S. Pat. No.4,863,457), such as an implantable device, e.g., a mechanical reservoir,an implant, or a device comprised of a polymeric composition, areparticularly useful for administration of the adenoviral vector. Theadenoviral vector also can be administered in the form ofsustained-release formulations (see, e.g., U.S. Pat. No. 5,378,475)comprising, for example, gel foam, hyaluronic acid, gelatin, chondroitinsulfate, a polyphosphoester, such as bis-2-hydroxyethyl-terephthalate(BHET), and/or a polylactic-glycolic acid.

The dose of adenoviral vector administered to the mammal will depend ona number of factors, including the size and location of a tumor, theextent of any side-effects, the particular route of administration, andthe like. The dose ideally comprises a “therapeutically effectiveamount” of adenoviral vector, i.e., a dose of adenoviral vector whichkills cancer cells in the subject, usually to an extent that causesshrinkage or disappearance of a tumor, and which lessens or eradicatescancer symptoms. The treatment may be completely effective (i.e., thecancer is eliminated, the subject may go into remission) or partiallyeffective (tumor development may be slowed so that the life expectancyis increased and/or the quality of life of the subject is improved). Thedesired response can entail the death of cancer cells, tumor shrinkage,tumor eradication, prevention or elimination of metastases, and thelike.

The adenoviral vector may be provided as a single dose or in multipledoses ranging from 1×10⁸ to 1×10¹² vp or 1×10¹⁰ to 1×10¹⁴ vp perapplication. As will be recognized, viral particles (vp) totalinfectious and non-infectious Ad (which can be 25-100 fold more thanplaque forming units (pfu). For example, for direct injection, e.g.,intravenous, intracardiac, intraperitoneal, etc. (1×10⁸ to 1×10¹³ viralparticles); when in a microbubble, the maximum viral load in the bubblescan be used which will result in a more focused deliver to the tumor andits surrounding vasculature. The does will vary depending on a number offactors including the type of tumor and the administration route. Sincethe invention utilizes cancer-specific conditionally replicationcompetent viruses (Ad. 5/3-CTV; Ad.5/3CTV-M7), higher pfu and viralparticles may be used. In the GBM model, 1×10⁹ pfu have been used, butthis could be higher in humans with toxicity being the limiting factor(if it occurs). Higher doses may be tolerated in vivo, and with higherdoses, more effective therapy is anticipated with fewer administrations.In some applications, a single dose of adenoviral vector comprises atleast about 1×10⁶ plaque forming units (pfu) or an equivalent higheramount of viral particles (which is referred to as viral particle units;vp; which are greater than infectious pfu) of the adenoviral vector. Thedose typically is at least about 1×10⁸ pfu (e.g., about 1×10⁹-1×10¹²particles), more typically at least about 1×10⁷ pfu, more typically atleast about 1×10⁸ pfu (e.g., about 1×10⁹-1×10¹¹ particles), and mosttypically at least about 1×10⁹ pfu (e.g., about 1×10¹⁰-1×10¹² particles)of the adenoviral vector. The dose, in some applications, may desirablycomprises no more than about 1×10¹² pfu (1×10¹⁴ particles, or no morethan about 1×10¹³ particles, or no more than about 1×10¹² particles, orno more than about 1×10¹¹ particles, and or no more than about 1×10¹⁰particles).

The adenoviral vector desirably is administered in a composition,preferably a pharmaceutically acceptable (e.g., physiologicallyacceptable or compatible) composition, which comprises a carrier,preferably a pharmaceutically (e.g., physiologically acceptable) carrierand the adenoviral vector. Any suitable carrier can be used within thecontext of the invention, and such carriers are well known in the art.The choice of carrier will be determined, in part, by the particularsite to which the composition is to be administered and the particularmethod used to administer the composition. The composition canoptionally be sterile or sterile with the exception of the inventiveadenoviral vector.

Suitable formulations for the composition include aqueous andnon-aqueous solutions, isotonic sterile solutions, which can containanti-oxidants, buffers, and bacteriostats, and aqueous and non-aqueoussterile suspensions that can include suspending agents, solubilizers,thickening agents, stabilizers, and preservatives. The formulations canbe presented in unit-dose or multi-dose sealed containers, such asampoules and vials, and can be stored in a freeze-dried (lyophilized)condition requiring only the addition of the sterile liquid carrier, forexample, water, immediately prior to use. Extemporaneous solutions andsuspensions can be prepared from sterile powders, granules, and tabletsof the kind previously described. Preferably, the carrier is a bufferedsaline solution. More preferably, the adenoviral vector for use in theinventive method is administered in a composition formulated to protectthe expression vector from damage prior to administration. For example,the composition can be formulated to reduce loss of the adenoviralvector on devices used to prepare, store, or administer the expressionvector, such as glassware, syringes, or needles. To this end, thecomposition preferably comprises a pharmaceutically acceptable liquidcarrier, such as, for example, those described above, and a stabilizingagent selected from the group consisting of polysorbate 80, L-arginine,polyvinylpyrrolidone, trehalose, and combinations thereof. Use of such acomposition will extend the shelf life of the vector, facilitateadministration, and increase the efficiency of the inventive method.Formulations for adenoviral vector-containing compositions are furtherdescribed in, for example, U.S. Pat. Nos. 6,225,289, 6,514,943, U.S.Patent Application Publication 2003/0153065 A1, and International PatentApplication Publication WO 00/34444. A composition also can beformulated to enhance transduction efficiency. In addition, one ofordinary skill in the art will appreciate that the adenoviral vector canbe present in a composition with other therapeutic orbiologically-active agents. For example, factors that controlinflammation, immune system stimulators, other anticancer agents, andthe like.

As indicated above, the present invention inter alia provides thespecified agent for use in a method of treating cancer and/or killingcancer cells in a subject in need thereof. For the avoidance of doubt,in this aspect the present invention may provide the specified agent foruse as a medicament in the specified method. Further, the presentinvention may provide the specified agent as an active therapeuticingredient in the specified method. Further, the present invention mayprovide the specified agent for use in a method of treatment of thehuman or animal body by therapy, the method comprising the specifiedmethod.

Before exemplary embodiments of the present invention are described ingreater detail, it is to be understood that this invention is notlimited to particular embodiments described, as such may, of course,vary. It is also to be understood that the terminology used herein isfor the purpose of describing particular embodiments only, and is notintended to be limiting, since the scope of the present invention willbe limited only by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges and are also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, representativeillustrative methods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention. Further, the dates ofpublication provided may be different from the actual publication dateswhich may need to be independently confirmed.

It is noted that, as used herein and in the appended claims, thesingular forms “a”, “an”, and “the” include plural referents unless thecontext clearly dictates otherwise. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentinvention. Any recited method can be carried out in the order of eventsrecited or in any other order, which is logically possible.

EXAMPLES Example 1. Enhanced Prostate Cancer Gene Transfer and TherapyUsing a Novel Serotype Chimera Cancer Terminator Virus (Ad. 5/3-CTV; Ad.5/3-CTV-M7) Abstract

Few options are available for treating patients with advanced prostatecancer (PC). As PC is a slow growing disease and accessible byultrasound, gene therapy could provide a viable option for thisneoplasm. Conditionally replication-competent adenoviruses (CRCAs)represent potentially useful reagents for treating prostate cancer (PC).We previously constructed a CRCA, Cancer Terminator Virus (CTV), whichshowed efficacy both in vitro and in vivo for PC. The CTV was generatedon a serotype 5-background (Ad.5-CTV; Ad.5-CTV-M7) with infectivitydepending on Coxsackie-Adenovirus Receptors (CARs). CARs are frequentlyreduced in many tumor types, including PCs thereby limiting effectiveAd-mediated therapy. Using serotype chimerism, a novel CTV (Ad.5/3-CTV;Ad.5/3-CTV-M7) was created by replacing the Ad.5 fiber knob with theAd.3 fiber knob thereby facilitating infection in a CAR-independentmanner. We evaluated Ad.5/3-CTV (Ad.5/3-CTV-M7) in comparison withAd.5-CTV (Ad.5-CTV-M7) in low CAR human PC cells, demonstrating higherefficiency in inhibiting cell viability in vitro. Moreover, Ad.5/3-CTV(Ad.5/3-CTV-M7) potently suppressed in vivo tumor growth in a nude mousexenograft model and in a spontaneously induced PC that develops inHi-myc transgenic mice. Considering the significant responses in a PhaseI clinical trial of a non-replicating Ad.5-mda-7 in advanced cancers,Ad.5/3-CTV (Ad.5/3-CTV-M7) will exert improved therapeutic benefit in aclinical setting.

Introduction

Prostate Cancer (PC) is the most frequently diagnosed cancer and is thesecond leading cause of cancer death among men in the US (Damber andAus, 2008; Siegel et al., 2012). It is estimated that 238,590 new PCcases will be diagnosed in 2013 and 29,720 men will die of PC. Patientswith localized disease may be treated with surgery or radiation, whereasthe treatment options for patients with metastatic disease is purelypalliative. Current therapies include hormonal therapy, radiotherapy andcytotoxic chemotherapeutic agents (Siegel et al., 2012; Sternberg,2002). Although existing approaches are beneficial in men with variousstages of PC, the complications frequently associated with theseconventional treatment options diminish positive clinical outcomes.Consequently, more efficient and innovative treatments are mandatory,and genetic therapies represent promising approaches for the treatmentof this neoplasm.

Using subtraction hybridization combined with induction of cancer cellterminal differentiation, our laboratory cloned melanomadifferentiation-associated gene-7/interleukin-24 (mda-7/IL-24) (Jiang etal., 1995; Jiang et al., 1996), a novel member of the IL-10-relatedcytokine gene family (Dash et al., 2010a; Sarkar et al., 2002a; Sauaneet al., 2003; Wolk et al., 2002). Subsequent studies documented thatmda-7/IL-24 displays almost ubiquitous antitumor properties in vitro andin vivo, leading to its rapid entry into the clinic, where its safetyand clinical efficacy, when administered by adenovirus (Ad.mda-7; INGN241), was observed in a phase I clinical trial in humans with advancedcarcinomas and melanomas (Cunningham et al., 2005; Fisher et al., 2003;Fisher et al., 2007; Jiang et al., 1996; Lebedeva et al., 2007c; Tong etal., 2005; Lebedeva et al., 2007a). mda-7/IL-24 preferentially inducesapoptosis in cancer cells while exerting no discernible toxic effectstoward normal cells (Dash et al., 2011a; Dash et al., 2010a; Sarkar etal., 2002b; Sauane et al., 2008) and it also elicits potent “antitumorbystander activity” in distant cancer cells as a consequence ofautocrine and paracrine secretion of MDA-7/IL-24 (Dash et al., 2010b;Fisher, 2005; Lebedeva et al., 2007b; Sauane et al., 2008; Su et al.,2001a; Su et al., 2005a; Lebedeva et al., 2007a).

Since PC is generally a relatively slow-growing disease, it may requirerepeated gene therapy treatments, with single or multiple genes, overthe lifespan of the patient. Conditionally replication-competentadenoviruses (CRCAs) provide a potentially valuable reagent for genetherapy (Curiel and Fisher, 2012). Using subtraction hybridization wecloned a novel rodent gene, progression elevated gene-3 (PEG-3), in thecontext of tumor progression in transformed rat embryo cells (Su et al.,1997). PEG-3: (i) displays elevated expression as a function ofoncogenic transformation (by diverse oncogenes) (Su et al., 2000; Su etal., 1997) (ii) induces an aggressive cancer phenotype without promotingtransformation when expressed in normal cells (Su et al., 1999; Su etal., 2002) and (iii) the gene promoter (PEG-Prom) has been isolated andshown to display elevated expression in both rodent and human tumors(including PC), with negligible expression in normal cells (includinghuman prostate epithelium) (Bhang et al., 2011; Das et al., 2012; Sarkaret al., 2007b; Sarkar et al., 2006; Sarkar et al., 2008; Sarkar et al.,2005a; Sarkar et al., 2005b; Su et al., 2001b; Su et al., 2005b).Considering the cancer-specific expression aspects of the PEG-Prom, weconstructed a bipartite serotype 5 CRCA (called a Cancer TerminatorVirus, Ad.5-CTV Ad.5-CTV-M7) in which the expression of E1A and E1Bgenes of Ad, necessary for replication, is controlled by the PEG-Prom(Sarkar et al., 2007b; Sarkar et al., 2006; Sarkar et al., 2008; Sarkaret al., 2005a). This novel Ad.5-CTV (Ad.5-CTV-M7) also expressedmda-7/IL-24 from the E3 region (Ad.PEG-E1A-mda-7). The ability ofAd.5-CTV (Ad.5-CTV-M7) to infect and express MDA-7/IL-24 in PC cellsdepends on the presence of Coxsackie-Adenovirus Receptors (CAR) on theirsurface. Ad.5-CTV (Ad.5-CTV-M7) is capable of efficiently infecting highCAR receptor cells (such as DU-145) and expressing robust levels ofmda-7/IL-24, whereas infection is restricted and expression ofMDA-7/IL-24 is minimal in low CAR receptor cells, such as PC-3 (Dash etal., 2011b; Dash et al., 2010b).

An approach to circumvent the low efficiency of Ad.5 infection of tumorcells involves ‘tropism modification’ in which virus capsid proteinsthat normally associate with CAR are modified, permitting bothCAR-dependent and CAR-independent infectivity of tumor cells. Studiesusing various tumor cell types have shown that inclusion of theinfective type 3 Ad sequence within the Ad type 5 virus knob (Ad.5/3recombinant virus) promotes viral infectivity in tumor cells displayingreduced or no CAR expression (Azab et al., 2012; Dash et al., 2010b;Eulitt et al., 2011; Hamed et al., 2010; Park et al., 2011). It is worthnoting that Ad.5/3 also retains high infectivity in CAR-expressing tumorcells showing equal efficacy when compared with Ad.5, thereby providingan expanded range of utility for Ad.5/3, in both low and highCAR-expressing tumor cells.

In this Example 1, we constructed and evaluated the in vitro and in vivoefficacy in low and high CAR PCs of a novel tropism-modified CTV inwhich the virus capsid proteins that normally associate with CAR weremodified, Ad.5/3-CTV (Ad.5/3-CTV-M7), permitting CAR-independentinfectivity of tumor cells. In low CAR PC-3 cells Ad.5/3-CTV (Ad.5/3-CTV-M7) is more efficient than Ad.5-CTV (Ad.5-CTV-M7) in infectingtumor cells, delivering a transgene (mda-7/IL-24), expressingMDA-7/IL-24 protein and inducing cancer-specific apoptosis. In an invivo context, Ad.5/3-CTV (Ad.5/3-CTV-M7) is superior to the Ad.5-CTV(Ad.5-CTV-M7) in inhibiting in vivo tumor growth and exerting anantitumor ‘bystander’ effect in nude mouse human PC xenografts andAd.5/3-CTV (Ad.5/3-CTV-M7) potently suppresses PC development in animmunocompetent Hi-Myc transgenic mouse model of PC.

Materials and Methods Cell Lines, Culture Conditions, and ViabilityAssays.

DU-145 and PC-3 PC cells were obtained from the American Type CultureCollection and cultured as described (Lebedeva et al., 2003).Construction and characterization of PC-3 overexpressing Bcl-2,PC-3-Bcl-2, and control clones containing the neomycin vector, PC-3-Neo,were described previously (Lebedeva et al., 2003). Cell viability wasdetermined by standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays(Lebedeva et al., 2003). Cell cultures were routinely tested formycoplasma using a kit from Sigma (MP-0025) and only mycoplasma freecells were used for these studies.

Construction of Ad.5/3-CTV (Ad.5/3-CTV-M7).

The genome of Ad.5/3-PEG-E1-mda-7 was generated in three consecutivesteps (FIG. 1). 1) Homologous recombination of pAd.5/3 genomic plasmidwith pShuttlE3 plasmid containing the mda-7/IL-24 expression cassetteand kanamycin selection results in the pAd.5/3-E3-mda-7 genome. 2)pAd.5/3-E3-mda-7 was cut with Swa I to excise the kanamycin resistancegene. 3) The resultant pAd.5/3-E3-mda-7 plasmid was recombined withpShuttlE1 plasmid containing E1A and E1B genes under control of thePEG-3 promoter resulting in Ad.5/3-PEG-E1-mda-7 (Ad.5/3-CTV;Ad.5/3-CTV-M7) genomic plasmid. This plasmid was digested with Pac I torelease viral ITRs and transfected in A549 cells to rescue the CRCA,Ad.5/3-CTV (Ad. 5/3-CTV-M7).

Preparation of Whole-Cell Lysates and Western Blot Analyses.

Preparation of whole-cell lysates and Western blot analyses wereperformed as previously described (Sarkar et al., 2005b). The primaryantibodies used were anti-MDA-7/IL-24 (Gen Hunter Corporation),anti-EF1{acute over (α)} (1:1,000; mouse monoclonal; Millipore),anti-Mcl-1 (1:500; mouse monoclonal; Santa Cruz), anti-BiP/GRP78 (1:500;rabbit monoclonal; Santa Cruz), anti-GRP94 (1:1000; rabbit monoclonal;Sigma), and anti-PARP (1:1000; rabbit monoclonal; Cell Signaling).

Human Prostate Cancer Xenografts in Athymic Nude Mice.

PC-3-Bcl-2 cells (2×10⁶) were injected s.c. in 100 μL of 1:1 PBS andMatrigel in the left and right flanks of male athymic nude mice(NCRnu/nu, 6-8 weeks old, ˜20 gm body weight) (Sarkar et al., 2005a).After establishing palpable tumors of ˜100-mm³, requiring ˜7-10 days,intratumoral injections of different Ads were given only to the tumorson the left flank at a dose of 1×10¹⁰ viral particles in 100 μl. Theinjections were given twice a week for four weeks. A minimum of fiveanimals was used per experimental point. Tumor volume was calculatedusing the formula: δ/6×larger diameter×(smaller diameter)². At the endof the experiment, the animals were sacrificed, and the tumors wereremoved and weighed.

Hi-Myc Mice and Animal Husbandry Protocols.

The VCU Institutional Animal Care and Use Committee approved theexperimental protocol used in this study and the animals were cared forin accordance with institutional guidelines. This study used Hi-Myctransgenic mice in which prostate-specific expression of human c-Myc isdriven by the rat probasin promoter with two androgen response elements(ARR2/probasin promoter) (Ellwood-Yen et al., 2003). Mice were obtainedfrom the Mouse Repository of the National Cancer Institute Mouse Modelsof Human Cancer Consortium at NCI Frederick, Md., USA. Mouse-tail DNAwas isolated using the DNeasy Blood & Tissue Kit from QIAGEN (Valencia,Calif.) and subjected to a PCR-based screening assay for genotyping. Forgenotyping Hi-MYC mice, the upstream primer (located within the ARR2-PBpromoter), 5′-AAACATGATGACTACCAAGCTTGGC-3′ (SEQ ID NO: 4) and thedownstream primer (within the MYC cDNA sequence)5′-ATGATAGCATCTTGTTCTTAGTCTTTTTCTTAATAGGG-3′ (SEQ ID NO: 5) were used togenerate a PCR product of 177 base pairs.

Preparation of Microbubbles (MBs), Ultrasound (US) Platform,Ultrasound-Targeted Microbubble Destruction (UTMD) and BI-97C1(Sabutoclax).

Preparation of MBs followed by UTMD for delivery of mda-7/IL-24expressing Ads has been described previously (Dash et al., 2011a).Targeson (Targeson) custom synthesis US contrast agent (perfluorocarbonMBs, encapsulated by a lipid monolayer and poly(ethyleneglycol)stabilizer) were obtained. MBs were reconstituted in the presence orabsence of 1 ml of 1×10¹¹ viral particles of indicated Ads andunenclosed surface-associated Ads were treated with complement aspreviously described (Cianfriglia et al., 1999; Howard et al., 2006).For in vivo experiments, US exposure was achieved with a Micro-MaxxSonoSite (SonoSite) US machine equipped with the transducer L25 set at0.7 Mechanical Index, 1.8 MPa for 10 minutes. Mice were sedated in anIMPAQ6 anesthesia apparatus (VetEquip, Pleasanton, Calif.) that wassaturated with 3-5% isofluorane and 10-15% oxygen with the aid of aprecision vaporizer to deliver the appropriate amount of anesthetic andto induce anesthesia. For microbubble/Ad injection a 27-gauge needlewith a heparin lock was placed within a lateral tail vein foradministration of contrast material. The mice received injections of 100μl of MBs with Ads through the tail vein 8 times in the span of 4 weeks.Ultrasound (sonoporation) was performed with a SonoSite scanner(SonoSite) equipped with the transducer L25 set at 0.7 Mechanical Index,1.8 MPa for 10 min in the ventral side of mice in the prostatic area.BI-97C1 (Sabutoclax) was administered intraperitoneally at a dose levelof 3 mg/kg 3× a week for the duration of the study (total 12injections). Compounds dissolved in 500 μL of solvent (ethanol/CremophorEL/saline=10:10:80) were injected intraperitoneally. At the end of theexperiment, the Hi-Myc mice were sacrificed and the prostate wasdissected. The harvested prostate was preserved in neutral bufferedformalin at 4° C. before embedding in paraffin for immunohistochemicalanalysis.

Immunohistochemical Staining.

For immunohistochemical (IHC) analysis, formalin-fixed andparaffin-embedded specimens were sectioned 3-4-im thick. Sections weredeparaffinized, re-hydrated and then quenched in 3% H₂O₂ for 20 min.Sections were washed with PBS and blocked in PBS containing 1% BSA for20 min at 37° C. Monoclonal anti-MDA-7/IL-24 (1:200) was incubated for 3hr at room temperature and then washed 3× in PBS. Sections wereincubated with an avidin-biotin-peroxidase complex (Vectastain Elite ABCkit, Vector Laboratories) and then washed 2× in PBS. Theimmunoreactivity was determined using diaminobenzidine (DAB) as thefinal chromogen. Finally, sections were counterstained with Meyer'sHematoxylin, dehydrated through a sequence of increasing concentrationsof alcohol, cleared in xylene and mounted with epoxydic medium. Sectionswere also processed for hematoxylin and eosin (H&E) staining.

Determination of Apoptotic Cells by TUNEL Assay.

For TUNEL assays, we used the DeadEnd Colorimetric TUNEL Assay kit(Promega, Madison, Wis.) performed according to the manufacturer'sinstructions. Briefly, paraffin-embedded slides were deparaffinized andrehydrated. Pre-equilibrated slides were labeled with a labelingDNA-strand break solution containing a biotinylated nucleotide mix (60min at 37° C.). After several washes in 2×SSC and PBS, slides wereblocked with hydrogen peroxide (3-5 min at room temperature). Afterseveral washes in PBS, the slides were mounted with mounting solutionwith DAPI. Apoptotic cells on the slides were observed under an Olympusepifluorescence microscope (×10 magnification; Olympus, Center Valley,Pa.) in randomly chosen fields. For detection of apoptosis in atime-dependent manner in vitro, PC-3 cells were grown in microscopicslide culture chambers (BD Bioscience) and cells were treated withAd.5/3-CTV (Ad.5/3-CTV-M7) and BI-97C1 (Sabutoclax) after which thecells were fixed with 4% formaldehyde at the indicated time and TUNELassays were performed as per the manufacturer's instruction using anOlympus epifluorescence microscope (×10 magnification; Olympus, CenterValley, Pa.) (Dash et al., 2011a).

Statistical analysis.

Statistical analysis was done using student t test, followed by Fisher'sprotected least significant difference analysis. P<0.05 was consideredsignificant.

Results: Ad.5/3-CTV (Ad.5/3-CTV-M7) Displays Enhanced Mda-7/IL-24Expression and Inhibition of Cell Viability in Low CAR Prostate CancerCells

The scheme for constructing Ad.5/3-CTV (Ad.5/3-CTV-M7), in which viralreplication is controlled by the PEG-Prom and which also expressesmda-7/IL-24 in an Ad.5/3 background, is depicted in FIG. 1 and describedin detail in Materials & Methods. As controls we used Ad.5-vec(replication-incompetent empty Ad.5), Ad.5/3-vec(replication-incompetent empty Ad.5/3), Ad.5-PEG-E1A, in which viralreplication is controlled by the PEG-Prom in an Ad.5 background, andAd.5/3-PEG-E1A, in which viral replication is controlled by the PEG-Promin an Ad.5/3 background. We compared MDA-7/IL-24 expression uponinfection of Ad.5/3-CTV (Ad.5/3-CTV-M7) and Ad.5-CTV (Ad.5-CTV-M7) in PCcells that contain low or high CAR on their surface. For this purpose,we used PC-3, which have a reduced level of CAR (D value 0.32) incomparison with DU-145, which express a high level of CAR (D value 0.92)(Dash et al., 2010b; Lebedeva et al., 2003). In PC-3, MDA-7/IL-24expression was significantly higher upon infection with Ad.5/3-CTV(Ad.5/3-CTV-M7), as compared to Ad.5-CTV (Ad.5-CTV-M7), whereasinfection with both Ad.5/3-CTV (Ad.5/3-CTV-M7) and Ad.5-CTV(Ad.5-CTV-M7) in DU-145 resulted in comparable expression of MDA-7/IL-24protein (FIGS. 2A and 2B). Infection with control Ad vectors did notresult in MDA-7/IL-24 expression. These findings indicate that infectionwith Ad.5/3-CTV (Ad.5/3-CTV-M7) promotes enhanced transgene delivery inlow CAR containing PC cells compared to Ad.5-CTV (Ad.5-CTV-M7), and bothCTVs are comparable in transgene delivery in high CAR PC cells. Ananalogous finding was evident when analyzing Ad replication bymonitoring E1A protein levels in PC-3 and DU-145 cells (FIGS. 2A and2B). In DU-145 cells a similar pattern of virus replication was apparentfollowing infection with Ad.5 or Ad.5/3 background viruses, while inPC-3 cells replication of Ad.5/3 was significantly elevated incomparison with infection with Ad.5 (FIGS. 2A and 2B).

The efficacy of Ad.5/3-CTV (Ad.5/3-CTV-M7) and Ad.5-CTV (Ad.5-CTV-M7) inreducing cell proliferation of PC cells was evaluated in vitro by MTTassays. Ad.5/3-CTV (Ad.5/3-CTV-M7) infection resulted in enhancedreduction in the viability of PC-3 cells as compared to Ad.5-CTV(Ad.5-CTV-M7) infection at m.o.i.'s of 500 and 1000 VP/cell on day3-post and day 6-post infection (FIG. 2C). In DU-145 cells bothAd.5/3-CTV (Ad.5/3-CTV-M7) and Ad.5-CTV (Ad.5-CTV-M7) showed parallelefficiencies in reducing growth when assayed using equivalent viraltiters and evaluated at parallel time points (FIG. 2D). It should benoted that Ad.5-PEG-E1A and Ad.5/3-PEG-E1A was as effective as Ad.5-CTV(Ad.5-CTV-M7) and Ad.5/3-CTV (Ad.5/3-CTV-M7) in reducing cell viabilityin both cell lines indicating that the profound effect of Ad replicationin inhibiting cell viability might mask the in vitro growth inhibitoryeffects of mda-7/IL-24. In these contexts, in vivo evaluation of CTV ismandatory to confirm the ‘antitumor bystander’ effect exerted byMDA-7/IL-24.

Ad.513-CTV (Ad.5/3-CTV-M7), but not Ad.5-CTV (Ad.5-CTV-M7), Induces ERStress and Apoptosis, and Overcomes Therapy Resistance in PC-3-Bcl-2Tumor Cells

We next analyzed the expression of mda-7/IL-24-downstream genes andsignals that confer its tumor suppressor properties upon infection withAd.5/3-CTV (Ad.5/3-CTV-M7) and Ad.5-CTV (Ad.5-CTV-M7) in low CAR PC-3cells. Ad.5/3-CTV (Ad.5/3-CTV-M7) induces an ER stress response(unfolded-protein response) and we therefore determined the expressionlevels of ER-stress markers. In PC-3-Neo cells, the levels of BiP/GRP78and GRP94 were significantly higher upon infection with Ad.5/3-CTV(Ad.5/3-CTV-M7) as compared with Ad.5-CTV (Ad.5-CTV-M7). Ad.5/3-CTV(Ad.5/3-CTV-M7) also efficiently induced apoptosis as evidenced byincreased cleavage of PARP (FIG. 3A). It should be noted, that infectionwith the conditionally replication competent Ad.5/3, Ad.5/3-PEG-E1A,also induced a stress response, as did Ad.5/3-CTV (Ad.5/3-CTV-M7), asindicated by enhanced BiP/GRP78, GRP94 and PARP cleavage (FIG. 3A).However, this effect was not evident with Ad.5-PEG-E1A or Ad.5-CTV (Ad.5-CTV-M7) infection.

The Bcl-2 gene family plays a central role in PC and over expression ofBcl-2 gene family members confers resistance to specific cancertherapeutics (Lebedeva et al., 2003). In this context, we evaluated theefficacy of Ad.5/3-CTV (Ad.5/3-CTV-M7) vs. Ad.5-CTV (Ad.5-CTV-M7) inPC-3-Bcl-2 cells (PC-3 cells that stably over express Bcl-2), which areresistant to mda-7/IL-24-mediated killing (Lebedeva et al., 2003). Ascontrol, we used PC-3-Neo cells that are stably transformed with thesame vector expressing only the neomycin resistance gene and not theBcl-2 gene. In MTT assays, Ad.5-PEG-E1A and Ad.5-CTV (Ad.5-CTV-M7) wereless effective in inhibiting cell proliferation (viability) ofPC-3-Bcl-2, whereas PC3-Neo cells were sensitive to these viruses (FIGS.3B and 3C). Accordingly, the effect of Ad.5-CTV was more robust insuppressing growth of PC-3-Neo as compared to PC-3-Bcl-2 cells, whereasthe Ad.5/3-CTV (Ad.5/3-CTV-M7) displayed equivalent vigorous in vitroanti-proliferative activity in both cell types (FIGS. 3B and 3C). Thesefindings support the enhanced potential therapeutic application ofAd.5/3-CTV (Ad.5/3-CTV-M7) vs. Ad.5-CTV (Ad.5-CTV-M7) in PC patientsfrequently showing Bcl-2 over expression and down-regulation of CAR.

Ad.5/3-CTV (Ad.5/3-CTV-M7) Eradicates Primary and Inhibits DistantPC-3-Bcl-2 Xenografts in Nude Mice

Experiments were performed to determine if the enhanced in vitroactivity of Ad.5/3-CTV (Ad.5/3-CTV-M7) compared to Ad.5-CTV(Ad.5-CTV-M7) in low CAR PC-3-Bcl-2 cells translates into enhanced invivo activity. PC-3-Bcl-2 tumor cells were inoculated in both the rightand left flanks of athymic nude mice. After ˜7 to 10 days palpable tumorxenografts of ˜100 mm³ developed and the mice received 8 intratumoralinjections only in the left flank tumors over a 4-week period with1×10¹⁰ viral particles per 100 μL. The Ads used for this study includedAd.5-vec, Ad.5/3-vec, Ad.5-PEG-E1A, Ad.5/3-PEG-E1A, Ad.5-CTV(Ad.5-CTV-M7) and Ad.5/3-CTV (Ad.5/3-CTV-M7). No injections wereadministrated to right flank tumors. PC-3-Bcl-2 formed large, aggressiveand actively proliferating tumors on both flanks that were not affectedby treatment with Ad.5-vec, Ad.5/3-vec, Ad.5-PEG-E1A or Ad.5-CTV(Ad.5-CTV-M7). Although Ad.5/3-PEG-E1A inhibited the growth of tumors onthe left flank, it had no effect on the distant tumors on the rightflank (FIGS. 4A, 4B, 4C and 4D). In contrast, Ad.5/3-CTV (Ad.5/3-CTV-M7)dramatically inhibited tumor growth on the injected left flank andmarkedly inhibited tumor growth on the right flank, exceeding thetherapeutic effect of any other viral treatment. These results providedefinitive evidence for enhanced therapeutic efficacy of Ad.5/3-CTV(Ad.5/3-CTV-M7) as compared to Ad.5-CTV (Ad.5-CTV-M7) in prostate tumorcells with reduced CAR, and highlight the potent ‘bystander anticancer’activity of mda-7/IL-24 resulting in growth inhibition of right-side,non-injected tumors. The effectiveness of Ad.5/3-CTV (Ad.5/3-CTV-M7) andAd.5-CTV (Ad.5-CTV-M7) in transducing MDA-7/IL-24 in vivo was confirmedby Western blotting using total protein extracts from the harvestedtumors and probing with MDA-7/IL-24 antibody. Ad.5/3-CTV (Ad.5/3-CTV-M7)generated more MDA-7/IL-24 protein in both flanks, validating thepreviously reported “bystander antitumor” effect of MDA-7/IL-24 (Chadaet al., 2004; Lebedeva et al., 2007a; Sarkar et al., 2007b; Su et al.,2005a; Su et al., 2001a). In contrast, only weak MDA-7/IL-24 proteinexpression was evident in Ad.5-CTV (Ad.5-CTV-M7) injected PC-3-Bcl-2tumor (left flank) and no protein expression was evident on the rightflank tumor (FIG. 4E). These findings demonstrate that in low CAR PCcells, Ad.5/3-CTV (Ad. 5/3-CTV-M7) can generate robust expression ofMDA-7/IL-24 protein that is sufficient to inhibit tumor cellproliferation, and exert ‘bystander antitumor’ activity mediated byMDA-7/IL-24 in distant tumors.

Combination Treatment of Ad.5/3-CTV (Ad.5/3-CTV-M7) and BI-97C1(Sabutoclax) Potentiates Inhibition of Prostate Tumor Growth In Vivo inImmune Competent Animals

Because PC is a relatively slow-growing disease, repeated systemic genetherapy applications in combination with anti-tumor chemotherapeuticagents over the life span of the patient may be necessary to provideenduring clinical responses (Damber and Aus, 2008; Di Lorenzo and DePlacido, 2006). Previous studies demonstrated that BI-97C1 (Sabutoclax),which is a pure optical derivative of Apogossypol (Wei et al., 2010),has significant activity as a single agent against PC cells in vitro andin vivo in nude mouse xenograft studies. Apogossypol derivativesantagonize the antiapoptotic Bcl-2 family members including Bcl-2 andMcl-1 (Wei et al., 2009). MDA-7/IL-24 induces cancer-specific apoptosisthrough the translational inhibition of Mcl-1 (Dash et al., 2010c).BI-97C1 (Sabutoclax) sensitizes prostate cancer cells tomda-7/IL-24-mediated toxicity in vitro and in vivo (Dash et al., 2011a).

Experiments were performed to determine if Ad.5/3-CTV (Ad.5/3-CTV-M7) incombination with BI-97C1 could also inhibit prostate tumor growth invivo. For this analysis we used an immunocompetent transgenic mousemodel of prostate cancer (the Hi-Myc mouse) that spontaneously developsPC. In Hi-Myc mice, prostate-specific c-Myc gene expression iscontrolled through the rat probasin promoter with two androgen responseelements (ARR2/probasin promoter). Hi-Myc mice develop prostaticintraepithelial neoplasia (mPIN) as early as 2 to 4 weeks of age andinvasive adenocarcinoma of the prostate at 6 months (Ellwood-Yen et al.,2003). Treatment was initiated at 22 weeks of age. The ability todeliver adenoviruses systemically is limited by sequestering of thevirus in the liver and clearance of the virus by the immune system(Koizumi et al., 2007; Muruve, 2004; Schenk et al., 2010). To overcomethese formidable problems we employed a microbubble-targeted ultrasounddestruction (UTMD) approach (Das et al., 2012; Greco et al., 2010;Howard et al., 2006; Lu et al., 2003) in which microbubblesincorporating adenoviruses are targeted to release therapeutic virusesat the tumor site using ultrasound. Using ultrasound to sonicate themicrobubbles creates transient nonlethal perforations in cells and othermembranes. In this way, systemic and targeted delivery of mda-7/IL-24 tothe prostate of Hi-Myc mice was performed by tail vein injection ofmicrobubbles (MB) incorporating Ad.5/3-vec, Ad.5/3-PEG-E1A or Ad.5/3-CTV(Ad.5/3-CTV-M7) followed by sonoporation in the prostatic area (Dash etal., 2011 a). A total of 8 tail vein injections of each adenovirus wereadministered over a 4-week period. BI-97C1 was administeredintraperitoneally (i.p.) in each group at 3 mg/kg 3× a week throughoutthe study. The prostates of Hi-myc mice were sectioned and stained forMDA-7/IL-24 and Ki-67, proliferation marker, and apoptosis induction wasanalyzed by TUNEL assay. MDA-7/IL-24 expression was accompanied byincreased TUNEL positive cells and decreased Ki-67 positive cells in theAd.5/3-CTV (Ad.5/3-CTV-M7) and BI-97C1-treated group compared to thecontrol groups (FIGS. 5A-C). Although, Ad.5/3-CTV (Ad.5/3-CTV-M7) aloneinduced significant apoptosis it was markedly augmented when used incombination with BI-97C1.

Discussion

The progression of PC is often slow and different therapeutics may berequired during various stages of this process and at multiple timesduring the life of the patient. In the context of gene therapy, it maybe necessary to employ different genes, used alone or in combination,and viral or non-viral gene delivery approaches over extended periods oftime (Dash et al., 2010a, 2011a, 2011b). Accordingly, the use ofconditionally replicating adenoviruses to administer therapeutic genesin prostate tumor cells represents a potentially viable treatment option(Sarkar et al., 2005a. 2005b, 2006, 2007, 2008; Curiel and Fisher, 2012;Das et al., 2012). A major challenge for effective gene therapy usingnon-replicating as well as conditionally replicating Ads is the abilityto specifically deliver nucleic acids directly into diseased tissue.Additionally, progress in gene therapy has been hampered by concernsover the safety and utility of viral vectors, particularly forintravenous delivery, and the inefficiency of currently availablenon-viral transfection techniques (Dash et al., 2011b; Curiel andFisher, 2012). Recombinant Ads are one of the most common gene transfervectors utilized in human clinical trials, but systemic administrationof this virus is thwarted by host innate and adaptive antiviral immuneresponses which can limit and/or preclude repetitive treatment regiments(Jiang et al., 2004; Curiel and Fisher, 2012). Systemic delivery of Adsis also restricted because of non-specific trapping in the liver,preventing virus from reaching the diseased cells disseminatedthroughout the body. We have identified a superior gene therapy approachthat employs a novel therapeutic gene mda-7/IL-24, a unique member ofthe IL-10 gene family of cytokines (Jiang et al., 1995, 1996; Fisher etal., 2003, 2007; Fisher, 2005; Lebedeva et al., 2005, 2007; Emdad etal., 2009; Dash et al., 2010a). Early phase I clinical studies suggestthat mda-7/IL-24 may be an ideal agent for gene therapy of advancedcancers, including carcinomas from multiple organs and melanomas (Fisheret al., 2003, 2007; Cunningham et al., 2005; Tong et al., 2005; Eager etal., 2008). mda-7/IL-24 selectively induces apoptosis or toxic autophagyin a broad spectrum of human cancer cells in vitro and in vivo in animalmodels, whereas it appears devoid of toxicity in diverse normal humancells (Jiang et al, 1996; Huang et al., 2001; Sarkar et al., 2002a,2005a, 2005b; Sauane et al., 2003, 2006, 2008; Fisher et al., 2003,2007; Fisher, 2005; Lebedeva et al., 2005, 2007; Cunningham et al.,2005; Tong et al., 2005; Bhutia et al., 2010; Das et al., 2010a; Hamedet al., 2010b). Additionally, MDA-7/IL-24 is a secreted cytokine thatexhibits potent direct and indirect “bystander antitumor” effects onadjacent and distant cancer cells (Su et al., 2001b, 2005a; Wolk et al.,2002; Chada et al., 2004; Fisher, 2005; Dash et al., 2010a).

The ability of type 5 Ad (Ad.5) to infect mammalian cells is dependenton the level of CAR on the cell surface. This limitation and correlationbetween infectivity and levels of CAR expression in human PC cells hasbeen demonstrated previously (Okegawa et al., 2000; Pandha et al., 2003;Curiel and Fisher, 2012). DU-145 has high CAR, while PC-3 has lower cellsurface CAR expression, which makes it relatively resistant to Ad.5infection. To overcome resistance of PC-3 to Ad.5 infection, PC-3 cellshave been genetically engineered to increase the original 35% CAR foundin low CAR-positive cells to 86% CAR-positive cells (Okegawa et al.,2000). Consequently, infection with a recombinant Ad.5-CMV-p21virusresulted in higher levels of p21 protein in the genetically altered PC-3(enhanced CAR positive) virus-infected cells, thereby resulting inapoptosis (Okegawa et al., 2000). These studies highlight the importanceof CAR as a major determinant of Ad.5-based gene therapy approaches.

To enhance therapeutic efficacy of Ad gene therapy we have used a numberof approaches. We constructed a bipartite Ad.5 where viral replicationis controlled by the minimal active region of the promoter of the PEG-3gene (Su et al., 1997, 2005b), restricting viral replication to cancercells with limited activity in normal cells, and mda-7/IL-24 is drivenby a CMV promoter from the E3 region of Ad.5 (Sarkar et al., 2005a,2006, 2007, 2008). These viruses, termed Cancer Terminator Viruses(CTVs) (reviewed in Das et al., 2012), have shown profound activity inathymic nude mouse human xenograft models, including breast carcinomas,prostate cancer (including therapy-resistant prostate cancers overexpressing Bcl-2 or Bcl-X_(L)) and metastatic melanoma (Sarkar et al.,2005a, 2006, 2007, 2008; Greco et al., 2010). The enhanced therapeuticactivity of the CTV relates to the profound direct apoptosis- and toxicautophagy-inducing effects and indirect antitumor activity of thissecreted cytokine through its “bystander” effects, which includeinhibition of tumor angiogenesis, synergism with other modes of cancertherapy (including chemotherapy, radiation and monoclonal antibodies)and promotion of a potent immune response against the tumor (Fisher,2005; Lebedeva et al., 2007; Sarkar et al., 2007; Gao et al., 2008).

To enhance viral entry into cancer cells, many of which showdownregulation of CAR, we engineered chimeric adenoviruses containingthe Ad.3 sequence within the Ad.5 virus knob (Ad.5/3) (Dash et al.,2010b; Azab et al., 2012), which redirects binding of the vector to theAd.3 receptor, desmoglein 2 (Wang et al., 2011). The finding thatAd.5/3-CTV (Ad. 5/3-CTV-M7) eradicated not only primary injected tumors,but also distant non-injected tumors derived from a resistant PC cellline in a nude mouse xenograft model support the anticancer potency ofthis cancer therapeutic virus. As discussed, Ad.5/3-CTV (Ad.5/3-CTV-M7)is capable of infecting cancer cells regardless of their cell surfaceCAR status, which makes it potentially more efficacious than Ad.5-CTV(Ad.5/3-CTV-M7) that fails to efficiently infect and deliver thetherapeutic genes in low CAR PC cells.

Systematic delivery of Ads is challenging because of sequestration ofviruses in the liver restricting efficient delivery to disseminatedtumors (Koizumi et al., 2007) and neutralization of viruses by theimmune system (Koizumi et al., 2007). To prevent trapping of CTV in theliver and elimination of viruses by the immune system we have developedan innovative approach that involves the use of perfluorocarbonmicrobubbles and ultrasound (Greco et al., 2010; Dash et al., 2011a,2011b). This approach is called ultrasound-targetedmicrobubble-destruction (UTMD). We have applied the UTMD approach usinga tropism-modified Ad.5/3-CTV in Hi-Myc transgenic mice, which developPC (Ellwood-Yen et al., 2003; Dash et al., 2011a). Ad.5/3-CTV(Ad.5/3-CTV-M7) in complement-treated microbubbles were administeredsystemically through the tail vein of mice and released in the prostatearea through ultrasound in this syngeneic immunocompetent prostatecancer mouse model (Hi-Myc mouse) using UTMD (Dash et al., 2011a,2011b). Hi-Myc mice develop, with high penetrance, prostaticintraepithelial neoplasia (PIN) that advances over time to invasiveadenocarcinomas in all lobes of the prostate gland (Ellwood-Yen et al.,2003). In the present study show the combinatorial anticancer effect ofAd.5/3-CTV (Ad.5/3-CTV-M7) and the novel Mcl-1 antagonist, BI-97C1(Sabutoclax), which significantly inhibits PC in Hi-Myc transgenic mice.For combination studies we chose an Mcl-1 antagonist based on ourprevious observations where we demonstrated that suppression of thepro-survival Bcl-2 family member, myeloid cell leukemia-1 (Mcl-1), isrequired for mda-7/IL-24-mediated apoptosis of prostate carcinomas (Dashet al., 2010c; Dash et al., 2011a). Here we demonstrate thatpharmacological inhibition of Mcl-1 expression with the novelApogossypol derivative BI-97C1 is sufficient to sensitize prostatetumors to mda-7/IL-24-induced (Ad.5/3-CTV) apoptosis.

In summary,

1) The prostate gland is not vital for survival and it is accessible byultrasound.

2) Ad.5/3-CTV (Ad.5/3-CTV-M7) can be either injected directly into theprimary tumor or delivered through UTMD with complement-treatedmicrobubbles incorporating Ads resulting in cancer cell lysis andexpression of MDA-7/IL-24 when ultrasound is applied.

3) The replication of this virus and the expression of the therapeuticgenes can be targeted in cancer cells without non-specific expression innormal cells by using the cancer-specific and tissue-specific PEG-3promoter.

4) Disease progression can be effectively monitored by measuringprostate-specific antigen (PSA) (Cookson, 2001; Gopalkrishnan et al.,2001). In these contexts, the use of Ad.5/3-CTV (Ad.5/3-CTV-M7) toadminister the therapeutic and cancer-specific cytotoxic mda-7/IL-24protein to selectively induce cytolysis and apoptosis in prostate tumorcells represents a potentially viable treatment option (Anderson, 1998;Sarkar et al., 2007; Das et al., 2012).

REFERENCES FOR EXAMPLE 1

-   Anderson, W F. 1998. Human gene therapy. Nature 392: 25-30.-   Azab, B, Dash, R, Das, S K, Bhutia, S K, Shen, X N, Quinn, B A,    Sarkar, S, Wang, X Y, Hedvat, M, Dmitriev, I P, Curiel, D T, Grant,    S, Dent, P, Reed, J C, Pellecchia, M, Sarkar, D, and Fisher,    P B. 2012. Enhanced delivery of mda-7/IL-24 using a serotype    chimeric adenovirus (Ad.5/3) in combination with the Apogossypol    derivative BI-97C1 (Sabutoclax) improves therapeutic efficacy in low    CAR colorectal cancer cells. J Cellular Physiol, 227: 2145-2153.-   Bhang, H-e, Gabrielson, K L, Laterra, J, Fisher, P B, and Pomper,    M G. 2011. Tumor-specific imaging through progression elevated    gene-3 promoter-driven gene expression. Nature Med 17: 123-129.-   Bhutia, S K, Dash, R, Das, S K, Azab, B, Su, Z Z, Lee, S G, Grant,    S, Yacoub, A, Dent, P, Curiel, D T, Sarkar, D, and Fisher,    P B. 2010. Mechanism of autophagy to apoptosis switch triggered in    prostate cancer cells by antitumor cytokine melanoma    differentiation-associated gene 7/interleukin-24. Cancer Res 70:    3667-3676.-   Chada, S, Mhashilkar, A M, Ramesh, R, Mumm, J B, Sutton, R B,    Bocangel, D, Zheng, M, Grimm, E A, and Ekmekcioglu, S. 2004.    Bystander activity of Ad-mda7: human MDA-7 protein kills melanoma    cells via an IL-20 receptor-dependent but STAT3-independent    mechanism. Mol Ther 10: 1085-1095.-   Cookson, M M. 2001. Prostate cancer: screening and early detection.    Cancer Control 8: 133-140.-   Cunningham, C C, Chada, S, Merritt, J A, Tong, A, Senzer, N, Zhang,    Y, Mhashilkar, A, Parker, K,-   Vukelja, S, Richards, D, Hood, J, Coffee, K, and    Nemunaitis, J. 2005. Clinical and local biological effects of an    intratumoral injection of mda-7 (IL24; INGN 241) in patients with    advanced carcinoma: a phase I study. Mol Ther 11: 149-159.-   Curiel D T, and Fisher P B. 2012. Applications of viruses for cancer    therapy. Tew K D and Fisher P B (series editors). Adv Cancer Res    115: 1-334.-   Damber J E, AG. 2008. Prostate cancer. Lancet 371: 1710-1721.-   Dash, R, Bhutia, S K, Azab, B, Su, Z z, Quinn, B A, Kegelmen, T P,    Das, S K, Kim, K, Lee, S G, Park, M A, Yacoub, A, Rahmani, M, Emdad,    L, Dmitriev, I P, Wang, X Y, Sarkar, D, Grant, S, Dent, P, Curiel, D    T, and Fisher, P B. 2010a. mda-7/IL-24: a unique member of the IL-10    gene family promoting cancer-specific toxicity. Cytokine & Growth    Factor Rev 21: 381-391.-   Dash, R, Dmitriev, I, Su, Z Z, Bhutia, S K, Azab, B, Vozhilla, N,    Yacoub, A, Dent, P, Curiel, D T, Sarkar, D, and Fisher, P B. 2010b.    Enhanced delivery of mda-7/IL-24 using a serotype chimeric    adenovirus (Ad.5/3) improves therapeutic efficacy in low CAR    prostate cancer cells. Cancer Gene Ther 17: 447-456.-   Dash, R, Richards, J E, Su, Z Z, Bhutia, S K, Azab, B, Rahmani, M,    Dasmahapatra, G, Yacoub, A, Dent, P, Dmitriev, I P, Curiel, D T,    Grant, S, Pellecchia, M, Reed, J C, Sarkar, D, and Fisher, P B.    2010c. Mechanism by which Mcl-1 regulates cancer-specific apoptosis    triggered by mda-7/IL-24, an IL-10-related cytokine. Cancer Res 70:    5034-5045.-   Dash, R, Azab, B, Quinn, B A, Shen, X, Wang, X Y, Das, S K, Rahmani,    M, Wei, J, Hedvat, M, Dent, P, Dmitriev, I P, Curiel, D T, Grant, S,    Wu, B, Stebbins, J L, Pellecchia, M, Reed, J C, Sarkar, D, and    Fisher, P B. 2011a. Apogossypol derivative BI-97C1 (Sabutoclax)    targeting Mcl-1 sensitizes prostate cancer cells to    mda-7/IL-24-mediated toxicity. Proc Natl Acad Sci USA 108:    8785-8790.-   Dash, R, Azab, B, Shen, X N, Sokhi, U K, Sarkar, S, Su, Z Z, Wang, X    Y, Claudio, P P, Dent, P, Dmitriev, I P, Curiel, D T, Grant, S,    Sarkar, D, and Fisher, P B. 2011b. Developing an effective gene    therapy for prostate cancer: new technologies with potential to    translate from the laboratory into the clinic. Discov Med 11: 46-56.-   Di Lorenzo, G, and De Placido, S. 2006. Hormone refractory prostate    cancer (HRPC): present and future approaches of therapy. Int J    Immunopathol Pharmacol 19: 11-34.-   Ellwood-Yen, K, Graeber, T G, Wongvipat, J, Iruela-Arispe, M L,    Zhang, J, Matusik, R, Thomas, G V, and Sawyers, C L. 2003.    Myc-driven murine prostate cancer shares molecular features with    human prostate tumors. Cancer Cell 4: 223-238.-   Emdad, L, Lebedeva, I V, Su, Z-z, Gupta, P, Sauane, M, Dash, R,    Grant, S, Dent, P, Curiel, D T, Sarkar, D, and Fisher, P B. 2009.    Historical perspective and recent insights into our understanding of    the molecular and biochemical basis of the antitumor properties of    mda-7/IL-24. Cancer Biol & Ther 8: 391-400.-   Eulitt, P J, Park, M A, Hossein, H, Cruikshanks, N, Yang, C,    Dmitriev, I P, Yacoub, A, Curiel, D T, Fisher, P B, and    Dent, P. 2011. Enhancing mda-7/IL-24 therapy in renal carcinoma    cells by inhibiting multiple protective signaling pathways using    sorafenib and by Ad. 5/3 gene delivery. Cancer Biol Ther 10:    1290-1305.-   Fisher, P B. 2005. Is mda-7/IL-24 a ‘magic bullet’ for cancer?    Cancer Res 65: 10128-10138.-   Fisher, P B, Gopalkrishnan, R V, Chada, S, Ramesh, R, Grimm, E A,    Rosenfeld, M R, Curiel, D T, and Dent, P. 2003. mda-7/IL-24: A novel    cancer selective apoptosis inducing cytokine gene: From the    laboratory into the clinic. Cancer Biol Therapy 2: S23-S37.-   Fisher P B, Sarkar D, Lebedeva I V, Emdad L, Gupta P, Sauane M, Su    Z-z, Grant S, Dent P, Curiel D T, Senzer N, Nemunaitis J. 2007.    Melanoma differentiation associated gene-7/interleukin-24    (mda-7/IL-24): novel gene therapeutic for metastatic melanoma.    Toxicol & Applied Pharmacol 224: 300-307.-   Gao, P, Sun, X, Chen, X, Wang, Y, Foster, B A, Subjeck, J, Fisher, P    B, and Wang, X Y. 2008. Secretable chaperone Grp170 enhances    therapeutic activity of a novel tumor suppressor, mda-7/IL-24.    Cancer Res 68: 3890-3898.-   Gopalkrishnan, R V, Kang, D C, and Fisher, P B. 2001. Molecular    markers and determinants of prostate cancer metastasis. J Cell    Physiol 189: 245-256.-   Greco, A, Di Benedetto, A, Howard, C M, Kelly, S, Nande, R,    Dementieva, Y, Miranda, M, Brunetti, A, Salvatore, M, Claudio, L,    Sarkar, D, Dent, P, Curiel, D T, Fisher, P B, and Claudio,    P P. 2010. Eradication of Therapy-resistant Human Prostate Tumors    Using an Ultrasound-guided Site-specific Cancer Terminator Virus    Delivery Approach. Mol Ther 18: 295-306.-   Hamed, H A, Yacoub, A, Park, M A, Eulitt, P J, Dash, R, Sarkar, D,    Dmitriev, I P, Lesniak, M S, Shah, K, Grant, S, Curiel, D T, Fisher,    P B, and Dent, P. 2010a. Inhibition of multiple protective signaling    pathways and Ad. 5/3 delivery enhances mda-7/IL-24 therapy of    malignant glioma. Mol Ther 18: 1130-1142.-   Hamed, H A, Yacoub, A, Park, M A, Eulitt, P, Sarkar, D, Dmitriev, I    P, Chen, C-S, Grant, S, Curiel, D T, Fisher, P B, and Dent, P.    2010b. OSU-03012 enhances Ad.mda-7-induced GBM cell killing via ER    stress and autophagy and by decreasing expression of mitochondrial    protective proteins. Cancer Biol & Ther 9: 526-536.-   Howard, C M, Forsberg, F, Minimo, C, Liu, J B, Merton, D A, and    Claudio, P P. 2006. Ultrasound guided site specific gene delivery    system using adenoviral vectors and commercial ultrasound contrast    agents. J Cell Physiol 209: 413-421.-   Huang, E Y, Madireddi, M T, Gopalkrishnan, R V, Leszczyniecka, M,    Su, Z, Lebedeva, I V, Kang, D, Jiang, H, Lin, J J, Alexandre, D,    Chen, Y, Vozhilla, N, Mei, M X, Christiansen, K A, Sivo, F,    Goldstein, N I, Mhashilkar, A B, Chada, S, Huberman, E, Pestka, S,    and Fisher, P B. 2001. Genomic structure, chromosomal localization    and expression profile of a novel melanoma differentiation    associated (mda-7) gene with cancer specific growth suppressing and    apoptosis inducing properties. Oncogene 20: 7051-7063.-   Jiang, H, and Fisher P B. 2003. Use of a sensitive and efficient    subtraction hybridization protocol for the identification of genes    differentially regulated during the induction of differentiation in    human melanoma cells. Mol Cell Different 1: 285-299.-   Jiang, H, Lin, J J, Su, Z Z, Goldstein, N I, and Fisher, P B. 1995.    Subtraction hybridization identifies a novel melanoma    differentiation associated gene, mda-7, modulated during human    melanoma differentiation, growth and progression. Oncogene 11:    2477-2486.-   Jiang, H, Su, Z Z, Lin, J J, Goldstein, N I, Young, C S, and Fisher,    P B. 1996. The melanoma differentiation associated gene mda-7    suppresses cancer cell growth. Proc Natl Acad Sci U S A 93:    9160-9165.-   Jiang, H, Wang, Z, Serra, D, Frank, M M, and Amalfitano, A. 2004.    Recombinant adenovirus vectors activate the alternative complement    pathway, leading to the binding of human complement protein C3    independent of anti-ad antibodies. Mol Ther 10: 1140-1142.-   Koizumi, N, Yamaguchi, T, Kawabata, K, Sakurai, F, Sasaki, T,    Watanabe, Y, Hayakawa, T, and Mizuguchi, H. 2007. Fiber-modified    adenovirus vectors decrease liver toxicity through reduced IL-6    production. J Immunol 178: 1767-1773.-   Lebedeva, I V, Sarkar, D, Su, Z Z, Kitada, S, Dent, P, Stein, C A,    Reed, J C, and Fisher, P B. 2003. Bcl-2 and Bcl-x(L) differentially    protect human prostate cancer cells from induction of apoptosis by    melanoma differentiation associated gene-7, mda-7/IL-24. Oncogene    22: 8758-8773.-   Lebedeva, I V, Sauane, M, Gopalkrishnan, R V, Sarkar, D, Su, Z z,    Gupta, P, Nemunaitis, J, Cunningham, C, Yacoub, A, Dent, P, and    Fisher, P B. 2005. mda-7/IL-24: Exploiting cancer's Achilles' heel.    Mol Therapy 11: 4-18.-   Lebedeva, I V, Emdad, L, Su, Z Z, Gupta, P, Sauane, M, Sarkar, D,    Staudt, M R, Liu, S J, Taher, M M, Xiao, R, Barral, P, Lee, S G,    Wang, D, Vozhilla, N, Park, E S, Chatman, L, Boukerche, H, Ramesh,    R, Inoue, S, Chada, S, Li, R, De Pass, A L, Mahasreshti, P J,    Dmitriev, I P, Curiel, D T, Yacoub, A, Grant, S, Dent, P, Senzer, N,    Nemunaitis, J J, and Fisher, P B. 2007. mda-7/IL-24, novel    anticancer cytokine: focus on bystander antitumor,    radiosensitization and antiangiogenic properties and overview of the    phase I clinical experience (Review). Int J Oncol 31: 985-1007.-   Lu, Q L, Liang, H D, Partridge, T, and Blomley, M J. 2003.    Microbubble ultrasound improves the efficiency of gene transduction    in skeletal muscle in vivo with reduced tissue damage. Gene Ther 10:    396-405.-   Muruve, D A. 2004. The innate immune response to adenovirus vectors.    Hum Gene Ther 15: 1157-1166.-   Eager R, Harle L, Nemunaitis J. 2008. Ad-MDA-7; INGN 241: a review    of preclinical and clinical experience. Expert Opin Biol Ther 8:    1633-1643.-   Okegawa, T, Li, Y, Pong, R C, Bergelson, J M, Zhou, J, and Hsieh,    J T. 2000. The dual impact of coxsackie and adenovirus receptor    expression on human prostate cancer gene therapy. Cancer research    60: 5031-5036.-   Pandha, H S, Stockwin, L H, Eaton, J, Clarke, I A, Dalgleish, A G,    Todryk, S M, and Blair, G E. 2003. Coxsackie B and adenovirus    receptor, integrin and major histocompatibility complex class I    expression in human prostate cancer cell lines: implications for    gene therapy strategies. Prostate Cancer Prostatic Dis 6: 6-11.-   Park, M A, Hamed, H A, Mitchell, C, Cruickshanks, N, Dash, R,    Allegood, J, Dmitriev, I P, Tye, G, Ogretmen, B, Spiegel, S, Yacoub,    A, Grant, S, Curiel, D T, Fisher, P B, and Dent, P. 2011. A serotype    5/3 adenovirus expressing MDA-7/IL-24 infects renal carcinoma cells    and promotes toxicity of agents that increase ROS and ceramide    levels. Mol. Pharmacol 79: 368-380.-   Sarkar, D, Su, Z Z, Lebedeva, I V, Sauane, M, Gopalkrishnan, R V,    Dent, P, and Fisher, P B. 2002a. Mda-7 (IL-24): Signaling and    functional roles. BioTechniques, Oct. Suppl., 30-39.-   Sarkar, D, Su, Z Z, Lebedeva, I V, Sauane, M, Gopalkrishnan, R V,    Valerie, K, Dent, P, and Fisher, P B. 2002b. mda-7 (IL-24) Mediates    selective apoptosis in human melanoma cells by inducing the    coordinated overexpression of the GADD family of genes by means of    p38 MAPK. Proc Natl Acad Sci USA 99: 10054-10059.-   Sarkar, D, Su, Z Z, Vozhilla, N, Park, E S, Gupta, P, and Fisher,    P B. 2005a. Dual cancer-specific targeting strategy cures primary    and distant breast carcinomas in nude mice. Proc Natl Acad Sci USA    102: 14034-14039.-   Sarkar, D, Su, Z Z, Vozhilla, N, Park, E S, Randolph, A, Valerie, K,    and Fisher, P B. 2005b. Targeted virus replication plus    immunotherapy eradicates primary and distant pancreatic tumors in    nude mice. Cancer research 65: 9056-9063.-   Sarkar, D, Su, Z Z, and Fisher, P B. 2006. Unique conditionally    replication competent bipartite adenoviruses-cancer terminator    viruses (CTV): efficacious reagents for cancer gene therapy. Cell    Cycle 5: 1531-1536.-   Sarkar, D, Lebedeva, I V, Su, Z Z, Park, E S, Chatman, L, Vozhilla,    N, Dent, P, Curiel, D T, and Fisher, P B. 2007. Eradication of    therapy-resistant human prostate tumors using a cancer terminator    virus. Cancer Res 67: 5434-5442.-   Sarkar, D, Su, Z Z, Park, E S, Vozhilla, N, Dent, P, Curiel, D T,    and Fisher, P B. 2008. A cancer terminator virus eradicates both    primary and distant human melanomas. Cancer Gene Therapy 15:    293-302.-   Sauane, M, Gopalkrishnan, R V, Sarkar, D, Su, Zz, Lebedeva, I V,    Dent, P, Pestka, S, and Fisher, P B. 2003. Mda-7/IL-24: novel cancer    growth suppressing and apoptosis inducing cytokine. Cytokine and    Growth Factor Reviews 14: 35-51.-   Sauane, M, Gupta, P, Lebedeva, I V, Su, Z Z, Sarkar, D, Randolph, A,    Valerie, K, Gopalkrishnan, R V, and Fisher, P B. 2006.    N-glycosylation of MDA-7/IL-24 is dispensable for tumor    cell-specific apoptosis and “bystander” antitumor activity. Cancer    Res 66: 11869-11877.-   Sauane, M, Su, Z Z, Gupta, P, Lebedeva, I V, Dent, P, Sarkar, D, and    Fisher, P B. 2008. Autocrine regulation of mda-7/IL-24 mediates    cancer-specific apoptosis. Proc Natl Acad Sci USA 105: 9763-9768.-   Schenk, E, Essand, M, Bangma, C H, Barber, C, Behr, J P, Briggs, S,    Carlisle, R, Cheng, W S, Danielsson, A, Dautzenberg, I J, Dzojic, H,    Erbacher, P, Fisher, K, Frazier, A, Georgopoulos, L J, Hoeben, R,    Kochanek, S, Koppers-Lalic, D, Kraaij, R, Kreppel, F, Lindholm, L,    Magnusson, M, Maitland, N, Neuberg, P, Nilsson, B, Ogris, M, Remy, J    S, Scaife, M, Schooten, E, Seymour, L, Totterman, T, Uil, T G,    Ulbrich, K, Veldhoven-Zweistra, J L, de Vrij, J, van Weerden, W,    Wagner, E, and Willemsen, R. 2010. Clinical adenoviral gene therapy    for prostate cancer. Hum Gene Ther 21: 807-813.-   Siegel, R, DeSantis, C, Virgo, K, Stein, K, Mariotto, A, Smith, T,    Cooper, D, Gansler, T, Lerro, C, Fedewa, S, Lin, C, Leach, C,    Cannady, R S, Cho, H, Scoppa, S, Hachey, M, Kirch, R, Jemal, A, and    Ward, E. 2012. Cancer treatment and survivorship statistics, CA    Cancer J Clin 62: 220-241.-   Stemberg, C N. 2002. Highlights of contemporary issues in the    medical management of prostate cancer. Crit Rev Oncol Hematol 43:    105-121.-   Su, Z Z, Shi, Y, and Fisher, P B. 1997. Subtraction hybridization    identifies a transformation progression-associated gene PEG-3 with    sequence homology to a growth arrest and DNA damage-inducible gene.    Proc Natl Acad Sci USA 94: 9125-9130.-   Su, Z Z, Goldstein, N I, Jiang, H, Wang, M N, Duigou, G J, Young, C    S, and Fisher, P B. 1999. PEG-3, a nontransforming cancer    progression gene, is a positive regulator of cancer aggressiveness    and angiogenesis. Proc Natl Acad Sci USA 96: 15115-15120.-   Su, Z, Shi, Y, and Fisher, P B. 2000. Cooperation between API and    PEA3 sites within the progression elevated gene-3 (PEG-3) promoter    regulate basal and differential expression of PEG-3 during    progression of the oncogenic phenotype in transformed rat embryo    cells. Oncogene 19: 3411-3421.-   Su, Z, Shi, Y, Friedman, R, Qiao, L, McKinstry, R, Hinman, D, Dent,    P, and Fisher, P B. 2001a. PEA3 sites within the progression    elevated gene-3 (PEG-3) promoter and mitogen-activated protein    kinase contribute to differential PEG-3 expression in Ha-ras and    v-raf oncogene transformed rat embryo cells. Nucleic Acids Res 29:    1661-1671.-   Su, Zz, Lebedeva, I V, Gopalkrishnan, R V, Goldstein, N I, Stein, C    A, Reed, J C, Dent, P, and Fisher, P B. 2001b. A combinatorial    approach for selectively inducing programmed cell death in human    pancreatic cancer cells. Proc Natl Acad Sci USA 98: 10332-10337.-   Su, Z Z, Gopalkrishnan, R V, Narayan, G, Dent, P, and Fisher,    P B. 2002. Progression elevated gene-3, PEG-3, induces genomic    instability in rodent and human tumor cells. J Cell Physiol 192:    34-44.-   Su, Z, Emdad, L, Sauane, M, Lebedeva, I V, Sarkar, D, Gupta, P,    James, C D, Randolph, A, Valerie, K, Walter, M R, Dent, P, and    Fisher, P B. 2005a. Unique aspects of mda-7/IL-24 antitumor    bystander activity: establishing a role for secretion of MDA-7/IL-24    protein by normal cells. Oncogene 24: 7552-7566.-   Su, Z Z, Sarkar, D, Emdad, L, Duigou, G J, Young, C S, Ware, J,    Randolph, A, Valerie, K, and Fisher, P B. 2005. Targeting gene    expression selectively in cancer cells by using the    progression-elevated gene-3 promoter. Proc Natl Acad Sci USA 102:    1059-1064.-   Wang, H, Li, Z, Yumul, R, Lara, S, Hemminki, A, Fender, P, and    Lieber, A. 2011. Multimerization of adenovirus serotype 3 fiber knob    domains is required for efficient binding of virus to desmoglein 2    and subsequent opening of epithelial junctions. J Virol 85:    6390-6402.-   Tong A W, Nemunaitis J, Su D, Zhang Y, Cunningham C, Senzer N, Netto    G, Rich D, Mhashilkar A, Parker K, Coffee K, Ramesh R, Ekmekcioglu    S, Grimm E A, van Wart Hood J, Merritt J, and Chada S. 2005.    Intratumoral injection of INGN 241, a nonreplicating adenovector    expressing the melanoma-differentiation associated gene-7    (mda-7/IL24): biologic outcome in advanced cancer patients. Mol Ther    11: 160-172.-   Wei, J, Kitada, S, Rega, M F, Stebbins, J L, Zhai, D, Cellitti, J,    Yuan, H, Emdadi, A, Dahl, R, Zhang, Z, Yang, L, Reed, J C, and    Pellecchia, M. 2009. Apogossypol derivatives as pan-active    inhibitors of antiapoptotic B-cell lymphoma/leukemia-2 (Bcl-2)    family proteins. J Med Chem 52: 4511-4523.-   Wei, J, Stebbins, J L, Kitada, S, Dash, R, Placzek, W, Rega, M F,    Wu, B, Cellitti, J, Zhai, D, Yang, L, Dahl, R, Fisher, P B, Reed, J    C, and Pellecchia, M. 2010. BI-97C1, an optically pure Apogossypol    derivative as pan-active inhibitor of antiapoptotic B-cell    lymphoma/leukemia-2 (Bcl-2) family proteins. J Med Chem 53:    4166-4176.-   Wolk, K, Kunz, S, Asadullah, K, and Sabat, R. 2002. Cutting edge:    immune cells as sources and targets of the IL-10 family members? J    Immunol 168: 5397-5402.

Example 2. Chemoprevention Gene Therapy (CGT) of Pancreatic Cancer UsingPerillyl Alcohol and a Novel Chimeric Serotype Cancer Terminator VirusAbstract

Conditionally replication competent adenoviruses (Ads) that selectivelyreplicate in cancer cells and simultaneously express a therapeuticcytokine, such as melanoma differentiation associatedgene-7/Interleukin-24 (mda-7/IL-24), a Cancer Terminator Virus (CTV-M7),hold potential for treating human cancers. To enhance the efficacy ofthe CTV-M7, we generated a chimeric Ad.5 and Ad.3 modified fiberbipartite CTV (Ad.5/3-CTV-M7) that can infect tumor cells in a CoxsackieAdenovirus receptor (CAR) independent manner, while retaining highinfectivity in cancer cells containing high CAR. Although mda-7/IL-24displays broad-spectrum anticancer properties, pancreatic ductaladenocarcinoma (PDAC) cells display an intrinsic resistance tomda-7/IL-24-mediated killing due to an mda-7/IL-24 mRNA translationalblock. However, using a chemoprevention gene therapy (CGT) approach withperillyl alcohol (POH) and a replication incompetent Ad to delivermda-7/IL-24 (Ad.mda-7) there is enhanced conversion of mda-7/IL-24 mRNAinto protein resulting in pancreatic cancer cell death in vitro and invivo in nude mice containing human PDAC xenografts. This combinationsynergistically induces mda-7/IL-24-mediated cancer-specific apoptosisby inhibiting anti-apoptotic Bcl-xL and Bcl-2 protein expression andinducing an endoplasmic reticulum (ER) stress response through inductionof BiP/GRP-78, which is most evident in chimeric-modifiednon-replicating Ad.5/3-mda-7- and Ad.5/3-CTV-M7-infected PDAC cells.Moreover, Ad.5/3-CTV-M7 in combination with POH sensitizestherapy-resistant MIA PaCa-2 cell lines over-expressing either Bcl-2 orBcl-xL to mda-7/IL-24-mediated apoptosis. Ad.5/3-CTV-M7 plus POH alsoexerts a significant antitumor ‘bystander’ effect in vivo suppressingboth primary and distant site tumor growth, confirming therapeuticutility of Ad.5/3-CTV-M7 plus POH in PDAC treatment, where all othercurrent treatment strategies in clinical settings show minimal efficacy.

Introduction

Pancreatic cancer is the fourth most common cause of cancer deaths inthe USA [1] and worldwide. This disease develops in an asymptomaticmanner, and is usually advanced or metastatic in >80% cases at the timeof diagnosis, making curative therapy impossible and leading to poorprognosis with incidence equaling mortality [1]. The plethora ofmolecular changes associated with progression of pancreatic cancer mayfacilitate its resistance to conventional chemotherapy and radiotherapy[2]. For this reason, it is imperative to develop rationalmolecular-targeted therapies that uniquely affect cancer cellsirrespective of the precise genetic alterations promoting the cancerousstate of these tumors. Obtaining this objective is particularly relevantin the context of pancreatic cancer.

In principle, conditionally replication competent Ads (CRCAs) shouldprovide a viable approach for cancer therapy, which has already beenapproved by the FDA in China to treat diverse cancers [3]. In mostcurrently used CRCAs, replication is dependent on a cells' p53 or pRbstatus, thereby limiting its universal applicability in cancer therapy.To create a more ubiquitous cancer-specific replicating CRCA, weengineered a CRCA manifesting the unique properties of tumor-specificE1A expression, thereby regulating virus replication, under the controlof the minimal promoter of rat progression elevated gene-3 (PEG-Prom)[4, 5] with concomitant production of mda-7/IL-24, referred to as aCancer Terminator Virus (CTV; Ad.PEG-E1A-mda-7; Ad.5-CTV-M7) [6, 7]. Inaddition to its cancer specific antitumor activity, mda-7/IL-24, anIL-10 gene family member secreted cytokine [8], can auto-regulate itsown production [9] making it a suitable candidate for gene therapy withpotential to eradicate not only primary infected tumor cells thatreceive mda-7/IL-24, but also distant tumor cells [10].

In the context of pancreatic cancer, decreased infectivity is asignificant obstacle for successful gene therapy using an Ad serotype-5(Ad.5) virus. The initial CTVs consisted of an Ad.5 expressingmda-7/IL-24 (Ad.5-CTV-M7) [6, 7] or interferon gamma (Ad.5-CTV-Q) [11],with infectivity depending on Coxsackie-Adenovirus Receptors (CAR) ontarget cells. The number of cell surface CAR is frequently reduced inmany tumor types, including pancreatic cancer [12], thereby limitingeffective therapy. Using serotype chimerism, a novel CTV expressingmda-7/IL-24 (Ad.5/3-CTV-M7) was created by replacing the Ad.5 fiber knobwith the Ad.3 fiber knob thereby facilitating infection in aCAR-independent manner [2, 13, 14]. An additional advantage of theAd.5/3-CTV-M7 is its retention of high infectivity of cancer cells thatcontain high CAR. We previously compared Ad.5/3-mda-7 with Ad.5-mda-7 inlow CAR PC-3 human prostate cancer cells, demonstrating higherefficiency in inhibiting cell viability in vitro [14]. Moreover,Ad.5/3-mda-7 potently suppressed in vivo tumor growth in a nude mousexenograft model and in spontaneously induced prostate cancer thatdevelops in Hi-myc transgenic mice [14]. Additional attributes ofmda-7/IL-24 as a cancer gene therapeutic agent, include its ability as asecreted cytokine to kill cancer cells at distant sites in the bodythrough “bystander” anticancer activity, which includes directapoptosis-induction of cancer cells, robust anti-angiogenic activity,potent immune modulating properties and an ability to synergize withconventional therapies, including radiation, chemotherapy andantibody-based therapy [15-18]. Based on the noteworthy activity ofAd.5/3-mda-7 [8, 9, 19, 20] and the Ad.5-CTV-M7 [6, 7] as anticancergene therapy agents, we anticipate that an Ad.5/3-CTV-M7 will provideeven more vigorous and wide spectrum antitumor activity [21].

Initial Phase I studies with Ad.mda-7 (INGN 241) injected directly intotumors of patients with advanced carcinomas and melanomas, demonstratedsafety and clinical activity in inducing cancer-specific apoptosis[22-26]. Unlike direct tumor administration of therapeutic viruses,systemic administration of viruses has generally proven disappointingand ineffective, particularly in the context of metastatic disease [27].Two significant impediments to effective systemic use of therapeuticviruses are non-specific trapping of viruses in the liver or othernon-tumor sites and neutralization and clearance of viruses by theimmune system [28]. To circumvent these obstacles, we are employing aninventive novel stealth-delivery approach to deliver encapsulated Ads inmicrobubbles (MBs) (Ad/MB) coupled with ultrasound targeted MBdestruction (ultrasound-targeted microbubble-destruction; UTMD)technology [2, 14, 29-31]. The effectiveness of the UTMD approach hasbeen confirmed by administering Ad.5-CTV-M7 in MBs resulting in curingof primary-treated and distant-untreated human prostate cancerxenografts in nude mice [29] and by delivering Ad.5/3-mda-7 in MBs inthe prostate region thereby decreasing prostate tumor volume and mass inHi-myc transgenic mice [14, 31].

Although effective in a wide gamut of genetically diverse human cancersin vitro and in vivo, pancreatic cancer cells display an intrinsicresistance towards mda-7/IL-24-induced killing [15]. This resistance ismanifested by a limited conversion of mda-7/IL-24 mRNA into protein inpancreatic cancer cells due to reduced association of this mRNA withpolysomes [32]. We demonstrated that ROS inducers (such as arsenictrioxide and dithiophene) could relieve this ‘translational block’ andsensitize both mutant and wild type K-RAS pancreatic cancer cells tomda-7/IL-24-induced apoptosis, which could be abrogated by ROSinhibitors (N-acetyl-L-cysteine or Tiron) [2, 32, 33]. ROS generation byhypoxia [34, 35] or UV-B [36], as well as extracellular H₂O₂[37],promotes phosphorylation of p70S6 Kinase and its downstream molecule4EBP-1, thereby activating the mTOR signaling pathway. Based on theseobservations, and our previous findings [32, 38], we predicted that ROSgenerated by perillyl alcohol (POH) might promote the formation ofpre-initiation complexes, resulting in the enhanced association ofweakly-translated mda-7/IL-24 with polysomes, subsequently leading tomore MDA-7/IL-24 protein. When POH was combined with a CTV producingmda-7/IL-24 (CTV-M7), which produces greater quantities of mda-7/IL-24mRNA as compared to non-replicating Ad.mda-7, we anticipated a greaterproduction of MDA-7/IL-24 protein and a more profound antitumor effectin pancreatic cancer cells. Moreover, we hypothesized that systemicdelivery of a chimeric recombinant CTV producing mda-7/IL-24(Ad.5/3-CTV-M7) by MB coupled with the UTMD approach combined with thechemoprevention non-toxic dietary agent POH might be beneficial ineliciting an enhanced antitumor response in pancreatic cancers in vivo,rather than using either agent alone or single agents with complementarymechanisms of action. We presently provide confirmation of thishypothesis and demonstrate profound therapeutic activity of theAd.5/3-CTV-M7 with POH, CGT approach, for the therapy of humanpancreatic cancers.

Material and Methods Cell Lines and Generating Stable Clones

AsPC-1 and BxPC-3 cell lines were obtained from the ATCC, maintained inRPMI-1640 (GIBCO®, Invitrogen™, Auckland, NZ) supplemented with 10%Fetal Bovine Serum (FBS) (Sigma-Aldrich, St. Louis, Mo., USA). MIAPaCa-2 and PANC-1, also obtained from the ATCC, were maintained in DMEM(GiBCO®) supplemented with 10% FBS, and Immortalized normal pancreaticmesenchymal cell line, LT-2 were obtained from EMD Millipore (Billerica,Mass., USA) and hTERT immortalized human pancreatic nestin expressingcells (hTERT-HPNE; HPNE-1) were obtained from the ATCC and weremaintained as instructed by the ATCC. All cell lines were cultured at37° C. in a 5% CO₂ and 95% air-humidified incubator. To obtain Bcl-2 andBcl-xL overexpressing clones, MIA PaCa-2 cells were transfected withpEGFP-C1/Bcl-2 (Addgene Plasmid 17999:GFP-Bcl-2) (Addgene, Cambridge,Mass., USA) or pSFFV-neo/Bcl-xL (Addgene Plasmid 8749: 3120 pSFFV-neoBcl-xL), respectively. Individual clones were selected after ˜3-4 weeksof continuous maintenance in culture medium containing 1 mg/ml G418sulfate [39], and were further characterized for the presence/expressionof the inserted plasmid by PCR and Western blotting.

Construction of Ad.5/3-PEG-E1A-Mda-7 (Ad-5/3-CTV-M7) To constructAd.5/3-PEG-E1A-mda-7, AdenoQuick cloning system (OD260, Inc., Boise,Id., USA) was employed. CMV-mda-7 along with Kan^(R) (kanamycinresistance marker) was inserted into the E1 deleted region of pAd.5/3(ΔE1) by homologous recombination between pAd5/3 (ΔE1) genomic plasmidwith pE3.1 shuttle vector containing the mda-7/IL-24 expression cassetteKanR, and Kanamycin selection strategy was used to selectAd.5/3-E3-E4-mda-7 and then pAd5/3.E3-E4-mda-7 was cut with Swa I toexcise the Kan^(R). The resultant pAd5/3.E3-mda-7 plasmid was recombinedwith the pE1.2 shuttle vector containing E1A-1B genes under control ofPEG-3 promoter resulting in Ad5/3.PEG-E1A-1B-mda-7 genomic plasmid. Thisplasmid was digested with Pac I to release viral ITRs and wastransfected into A549 cells to rescue the CRCA, Ad.5/3-PEG-E1A-E1B-mda-7(Ad.5/3-PEG-E1A-mda-7; Ad.5/3-CTV-M7). Similar strategies were employedto make Ad.5/3-mda-7 and the Ad.5/3-PEG-E1A construct. The constructswere purified using CsCl gradient, titrated both by OD260-SDS (vp/ml)(Optical absorbance at 260 nm of lysed Ad using 0.1% Sodiumdodecyl-sulphate solution) method and TCID50 (median or 50% tissueculture infective dose) or plaque forming methods (pfu/ml). We thankDrs. Curiel and Dmitriev (Washington University School of Medicine; St.Louis, Mo. USA for assistance in preparing and expanding various Ads.

Cell Viability Assays

Pancreatic cancer cell lines were infected with Ad in incomplete mediumfor 3 h followed by treatment with Perillyl alcohol (POH) (200 μM) incomplete medium (10% FBS) for 72 h, and cell proliferation assays wereperformed at O.D. 560 nm using cell proliferation assays after adding100 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(Sigma Aldrich) (MTT) dye (1 mg/ml) (Sigma-Aldrich).

Interaction Index or Combination Index

If agent 1 and agent 2 have 50% inhibitory effect, then it can beassumed that the combined effect of agent 1+agent 2 will be 0.5+0.5{(1-0.5)}=0.75 (75%). In this case the fraction effect for a combinationof two agents i.e., Fa(1,2) can be calculated from the single-agenteffects, i.e., F1 and F2, denoted by

Fa(1,2)=F1+F2(1−F1)  1 (Webb equation or fractional product method)

For mutually non-exclusive events, and considering the dose-effect graphis hyperbolic or the slope of the graph is 1, at each combination dose,Interaction index or combination index (CI) of two agents at given doseis denoted simply by,

$\begin{matrix}{{{CI} = \frac{{Fa}\left( {1,2} \right)}{{Foa}\left( {1,2} \right)}}{{{CI} < {1({Synergistic})}} = {{1({Additive})} < {1({Antagonistic})}}}} & 2\end{matrix}$

where F1, F2 are the fraction effects obtained from agent 1 and 2,respectively, at a given dose when used as single agents alone. Fa(1,2)is mathematically calculated fraction effect of two agents when used incombination at a fixed given dose and Foa(1,2) is the fraction effect oftwo agents obtained experimentally at the fixed given dose [40, 41].

Apoptotic Assays

Apoptotic assays were performed using an FITC Annexin V ApoptosisDetection Kit I (BD Pharmingen™, San Diego, Calif., USA), according tothe manufacturer's instructions. Flow cytometry assays were performedimmediately after staining using FACS Canto (BD Biosciencese). Data wereanalyzed using FACSDIVA software.

ROS Measurements

To determine ROS production, cells were stained with 100 μl of 10 μMcarboxy-H2DCFDA (Molecular Probes™, Invitrogen) in PBS for 30 minfollowed by treatment with Perillyl alcohol and fluorescence wasmeasured with a Fluorometer using a green filter at the indicated timepoints.

Preparation of Whole-Cell Lysates and Western Blotting Analyses

Cells were treated for 48 h, lysed using cell lysis buffer (CellSignaling Technology, Inc., Danvers, Mass., USA) supplemented with 1 mMPMSF (Sigma-Aldrich) with Protease cocktail inhibitor, Phosphataseinhibitor (Roche, Indianapolis, Ind. USA), and whole cell lysates werecollected after centrifugation at 12000 rpm at 4° C. For Westernblotting analyses, the primary antibodies used were mouse monoclonalanti-MDA-7/IL-24 (1:2000; Gen Hunter Corporation, Nashville, Tenn.,USA), anti-E1A (1:1000; EMD Millipore) anti-EF1{acute over (α)} (1:5000;EMD Millipore), rabbit monoclonal anti-Bcl-xL (1:1000), anti-PARP(1:1000), anti-Bcl-2 (1:1000; Cell Signaling Technology), rabbitpolyclonal anti-BiP/GRP-78 (1:500; Santa Cruz Biotechnology, Inc., SantaCruz, Calif., USA). The secondary antibodies used were polyclonal goatanti-mouse IgG (1:1000; Dako, Carpinteria, Calif., USA) and polyclonalswine anti-rabbit IgG (1:3000; Dako).

Detection of MDA-7/IL-24 Protein Using ELISA

MDA-7/IL-24 was quantified by using a human IL-24 DuoSet ELISADevelopment Kit from R&D Systems (R&D Systems, Inc., Minneapolis Mass.,USA) according to the manufacturer's instructions. The captureantibodies used were monoclonal mouse anti-human IL-24 (R&D Systems),and detection antibodies used were biotinylated conjugated goatanti-human IL-24 (R&D Systems), which was finally quantified byStreptavidin-HRP after adding substrate solution (H₂O₂ andTetramethylbenzidine). The cell culture supernatant or serum wascollected at the indicated times and stored at −80° C. until used forquantification. The absorbance was read at 450 nm with backgroundcorrections set at 560 nm.

Preparation of Adenoviruses-Complexed with Microbbubles (MB) (MB/Ad)

Perflurocarbon Microbbubles (MBs) encapsulated by lipid monolayer andpolyethylene glycol stabilizer were reconstituted in 1 ml of PBScontaining 1×10¹¹ viral particles of indicated Ads, and unenclosedsurface-associated Ads were treated with complement as previouslydescribed [42], and finally MB/Ad was dissolved in 1 ml of PBS prior totreatment.

In Vivo Xenograft Models

Athymic nude mice were injected s.c. in both flanks with 5×10⁶ MIAPaCa-2/luc (a stable clone of MIA PaCa-2 cells containing integratedpGL-3/luc). The mice were injected by the i.p. route daily with vehicleor POH (75 mg/kg body weight) dissolved in tricaprylin. When tumorsreached ˜100 mm³ in size (˜12-14 days), the animals were randomized intosubgroups (n=6 animals per subgroup) and MB/Ad were administered. Themice received injections of 100 μl of MB/Ad through the tail vein onceper week for a period of 4 weeks. Ultrasound (sonoporation) wasperformed with a SonoSite scanner (SonoSite) equipped with thetransducer L25 set at 0.7 Mechanical Index, 1.8 MPa for 10 min in theleft flank tumor of the of mice. Bioluminescence imaging (BLI) was doneusing a Xenogen In Vivo imaging system (IVIS) (Califer Life Sciences,Inc., Hopkinton, Mass.) after i.p. administration of D-luciferin (150mg/kg body weight). Images were analyzed by Living Image software. Atthe end of the experiment, the mice were sacrificed and tumors werecollected and preserved in neutral buffered formalin at 4° C. beforeembedding in paraffin for immunohistochemical analysis. The same tumorinduction and treatment protocol was followed for in vivo studies innude mice bearing MIA-PaCa-2/Bcl-xL (a stable clone of MIA PaCa-2 cellscontaining pSFFV-neo/Bcl-xL) xenograft model for demonstrating theefficacy of the CGT approach in therapy-resistant pancreatic cancers. Aminimum of six animals were used per experimental group. Tumor volume(since these cells did not contain a luciferase gene) was calculatedusing the formula: π/6×larger diameter×(smaller diameter)². At the endof the experiment, the animals were sacrificed, and the tumors wereremoved and weighed. The VCU Institutional Animal Care and Use Committeeapproved the experimental protocols used in this study and the animalswere cared for in accordance with institutional guidelines.

Statistical Analyses

Statistical analyses were performed using GraphPad Prism 5.0 (GraphPadSoftware, Inc.). Student's t-test or 1-way ANOVA was used as indicated,to study the level of significance (P<0.05).

Results

PEG-Prom is Selectively Expressed at Elevated Levels in PancreaticCancer Cells Vs. Normal Immortal Pancreatic Cells

The PEG-Prom, isolated from the PEG-3 gene [4, 5] displays selectivecancer-specific activity in a broad spectrum of cancers vs. normalcounterparts, including tumors of the prostate, breast, brain, colon,pancreas and melanoma [4, 6, 7, 11, 13, 31]. To confirm differentialexpression of the PEG-Prom in multiple pancreatic cancer cells lines,mutant K-ras pancreatic cancer cells, AsPC-1, PANC-1 and MIA PaCa-2 anda wild type K-ras pancreatic cancer cell, BxPC-3, and immortal normalpancreatic cells, LT-2 and HPNE-1, were infected withreplication-incompetent Ads expressing GFP or Luc under control of thePEG-Prom or CMV-Prom [4]. Cells were infected with 5000 vp/cell and wereexamined 2-days post infection. Ad.5-PEG-GFP activity was higher inpancreatic cancer cell lines vs. normal pancreatic cells as confirmed byimmunofluorescence (FIG. 6A) and flow-cytometric (FIG. 6B) analyses.Although MIA PaCa-2 expresses lower levels of CAR (unpublished data), itshowed high expression of Ad-PEG-GFP, since it expresses elevated levelsof AP-1, c-Jun [11]. The combination of AP-1 and PEA-3 transcriptionfactors is responsible for enhanced expression of the PEG-Prom indiverse cancers vs. normal cellular counterparts [4]. In order tominimize any differential effects due to CAR-dependent entry ofAd-5.GFP, we also infected parallel cultures with Ad.5-CMV-GFP, andnormalized the value of Ad.5-PEG-GFP/Ad.5-CMV-GFP (FIG. 6C). Aspredicted, GFP expression driven by the PEG-Prom displayed differentialelevated expression in the four pancreatic cancer cell lines. ThePEG-Prom activity of the PDAC cell lines MIA PaCa-2, PANC-1, AsPC-1 andBxPC-3 was significantly higher as compared to normal immortal pancreascounterparts, LT-2 and HPNE-1. To provide further confirmation ofPEG-Prom specificity and to avoid the dependence on receptors,especially CAR, for Ad entry, we also transfected the pancreatic cancercell lines with pPEG-luc and pRL-TK (20:1), and found that PEG-Promactivity was again significantly elevated in the PDAC cell lines ascompared to LT-2 and HPNE-1 cells (FIG. 6D).

Chimeric Modified Ad.5/3-CTV-M7 Potentiates the Activity of Mda-7/IL-24and Acts Synergistically with POH in Reducing Pancreatic Tumor CellViability

To test the hypothesis that bipartite CRCAs will cause oncolysis andreduce cell viability in PDAC as compared to normal pancreatic celllines, we engineered and employed a series of CRCAs with an Ad.5backbone (Ad.5-PEG-E1A, Ad.5-CTV-M7) as well as an Ad.5/3 backbone(Ad.5/3-mda-7, Ad.5/3-PEG-E1A and Ad.5/3-CTV-M7) in which thereplication of Ad is controlled by the PEG-Prom. Pancreatic cancer cellstreated with a replication incompetent Ad.5-mda-7 or Ad.5/3-mda-7 showedalmost no effect at an m.o.i. of 1000 vp/cell in all cell lines tested,whereas a minor effect was observed at a higher m.o.i. of 10000 vp/cellin the case of Ad.5/3-mda-7 after 3-days post-infection. With the CancerTerminator Virus (CTV-M7) cell viability was reduced dramatically evenat lower m.o.i. The IC₅₀ of PDAC cell lines treated with Ad.5-CTV-M7 wasfound to be ˜100 vp/cell in all cell lines tested with minimal effect inthe LT-2 cell line even at 10000 vp/cell of Ad.5-CTV-M7 (FIG. 13A-D).PDAC cell killing was most dramatic when cells were treated withAd.5/3-CTV-M7 (IC₅₀˜50 vp/cell).

Pancreatic cancer cells, containing either a wild type or mutant K-RASgene, display inherent resistance towards mda-7/IL-24-induced apoptosis[32], which can be reversed with addition of the chemoprevention agentPOH [38]. POH has been used to treat various solid malignancies and iscurrently undergoing evaluation in phase II clinical trials [2]. POH isvery unstable and cannot be detected in plasma and the major metabolitesof POH are Perillic acid and Dihydroperillic acid which have an ˜1-3 hhalf-life. The products are not accumulated over time even when POH isadministered at a dose of 800-1600 mg/m²/dose [43-45], which produceminimal side effects. Based on previous studies, 500 μM of Perillic acidin plasma was associated with minimal toxicities [43, 44]. Consideringthe toxicity profile, we choose a sub-lethal dose of 200 μM of POH,which is clinically achievable, to evaluate the efficacy of thischemoprevention dietary agent in potentially treating PDAC. Although 200NM of POH is not lethal and does not affect proliferation of PDAC celllines, this dose is sufficient to induce significant amounts of ROS in atemporal manner in both wild type and mutant K-ras pancreatic cell lines(FIG. 14). In previous studies [32], we did not notice appreciableformation of ROS after 24 h treatment with 200 μM POH, even though thistreatment promoted translation of the MDA-7/IL-24 protein and thiseffect was inhibited by addition of ROS inhibitors. Since we observed anincrease in ROS with treatment with 200 μM of POH at 6 h treatment, weperformed a time course study, which indicated elevated levels of ROSbetween 1 and 12 h, with maximum levels at 6 h and baseline levels ofROS at 24 h in PDAC cells following POH treatment (FIG. 14). Thistemporal relationship in ROS induction may explain the differences indetection of ROS in the present study as opposed to an earlier studymonitoring levels at 24 h [32]. It is also possible that the earlywindow of induction by POH is sufficient when enough mda-7/IL-24 mRNA isavailable to facilitate translation into MDA-7/IL-24 protein and deathin pancreatic cancer cells. In this context, the transient increase inROS by POH would be predicted to be more effective in producingMDA-7/IL-24 protein when infected with the CTV-M7, which results in highlevels of mda-7/IL-24 mRNA.

To investigate the potential combinatorial effect of our chemopreventiongene therapy (CGT) approach, both wild type K-ras (BxPC-3) and mutantK-ras (MIA PaCa-2) cells were infected with a replication incompetentAd.5-mda-7 or Ad.5/3-mda-7 (1-10000 vp/cell), or a CRCA eitherAd.5-CTV-M7 or Ad.5/3-CTV-M7, for 3 h followed by treatment with 200 μMPOH, and cell proliferation/viability was monitored 72 h post-infection.Combinatorial treatment with a replication incompetent Ad or CRCAcarrying mda-7/IL-24 with POH resulted in a leftward shift of the doseresponse curve in both cell lines indicating a synergistic effect ofthis combination (FIGS. 7A and 7B). Additionally, we mathematicallycalculated the effect of POH in combination with Ad.5-CTV-M7 andAd.5/3-CTV-M7. We found that there was a synergistic effect in both theBxPC-3 and MIA-PaCa-2 cell lines (FIGS. 15A-D).

Combinatorial Treatment with Ad Producing Mda-7/IL-24 and POH InducesApoptosis Induction and MDA-7/IL-24 Protein Production in PDAC CellLines

We next focused on defining the mechanism underlying reduced cellgrowth/viability and apoptosis induction in PDAC cells treated withreplication incompetent Ads (2500 vp/cell) or CRCAs (CTVs) (250 vp/cell)followed by POH treatment. Infection with Ads expressing mda-7/IL-24 andtreatment with POH significantly increased MDA-7/IL-24 proteinexpression, which correlated with its apoptotic activities as reflectedby PARP cleavage (FIG. 8). The cancer-specific replication of CRCAs, asconfirmed by E1A expression (FIG. 8), lead to elevated levels ofmda-7/IL-24 mRNA (FIGS. 9A, 9B, 9C and 9D) that might be responsible forincreased MDA-7/IL-24 protein expression in CTV-M7 as compared toMDA-7/IL-24 protein expression resulting from replication incompetentAds. By using 1 way-ANOVA, it was found that there were highlysignificant differences in mRNA expression in Ad.5-CTV-M7 andAd.5/3-CTV-M7 infected as compared to Ad.5-mda-7 infected PDAC cells(FIGS. 9C and 9D). Moreover, Ad replication leads to ROS [46], whichmight facilitate MDA-7/IL-24 production following infection with CTV-M7.

There was a decrease in Bcl-2 and Bcl-xL expression in MIA PaCa-2 andBxPC-3 cells following infection with Ad.5-CTV-M7 and Ad.5/3-CTV-M7,which decreased even further following combination therapy with POH(FIG. 8). No noticeable change was observed in either PDAC cell lineinfected with Ad.5-mda-7 or Ad.5/3-mda-7 alone, but the change in Bcl-2and Bcl-xL following CGT treatment correlated with intracellular as wellas extracellular/secreted MDA-7/IL-24 expression (FIGS. 8, 9A and 9B).MDA-7/IL-24 has Hsp-90-like chaperone (BiP/GRP-78) binding sites presentin the C and F helices, and mutations in these binding sites preventsmda-7/IL-24 from executing its apoptotic functions [47] in cancer cells.Binding of MDA-7/IL-24 to BiP/GRP-78 results in localization in theendoplasmic reticulum (ER) [32]. Since the ER is the principal site ofMDA-7/IL-24 localization and its subsequent folding; the increase inMDA-7/IL-24 expression can lead to an unfolded protein (UPR) responsethat results in increased production of chaperone binding proteinsBiP/GRP-78, GRP-94 and its downstream targets p38-MAPK and GADD thatconcludes in apoptosis in carcinomas of the breast, lung, prostate andmelanoma [9, 20, 47-49]. Ad.5-CTV-M7 and Ad.5/3-CTV-M7 infection of wildtype and mutant K-ras PDAC cell lines increase expression of BiP/GRP-78,which was further increased following treatment with POH as compared tountreated control Ad.5-vec and Ad.5/3-vec infected cells. Although therewas minimal or no expression of BiP/GRP-78 in Ad.5-mda-7 or Ad.5/3-mda-7infected cells, expression was increased dramatically followingcombination treatment, which coincided with the increased expression ofMDA-7/IL-24 that culminated in apoptosis, as reflected by PARP cleavageand Annexin V/PI staining (FIGS. 8 and 9A-F).

CTV-M7 Eradicates Therapy-Resistant Bcl-2 and Bcl-xL Overexpressing PDACCells

The antiapoptotic Bcl-2 family plays an important role in thedevelopment of therapy resistant pancreatic cancers [50]. Although Bcl-2plays a significant function in various cancers, it may not contributeto progression of pancreatic cancers, since expression decreases withPDAC progression and lymph node metastasis [51]. In contrast, Bcl-xLappears to be a key contributor to PDAC progression [52] andchemoresistance [50, 53]. Thapsigargin inhibits ER residing Ca⁺²-ATPasefunctions, and thus induces ER stress-mediated apoptosis [54].Downregulation of Bcl-xL sensitizes 0-islet cells toThapsigargin-induced ER-mediated apoptosis [54], whereas overexpressionof Bcl-xL protects cells from ER-mediated apoptosis [55, 56]. Bcl-2 andBcl-xL also protect prostate carcinoma cells from mda-7/IL-24-mediatedinduction of growth suppression and apoptosis [14, 39]. ER-stress playsan important role in mda-7/IL-24-mediated apoptosis in pancreaticcancers (FIGS. 8 and 9). Moreover, Bcl-xL is markedly downregulated bymda-7/IL-24 plus POH in PDAC cells. Thus, it is anticipated thatoverexpression of Bcl-xL might play a pivotal role in developingintrinsic resistance towards Ad.5/3-mda-7-mediated therapy, however, inthe presence of Ad.5-CTV-M7 or Ad.5/3-CTV-M7 and POH, robust expressionof MDA-7/IL-24 may lead to increased and sustained ER stress culminatingin PDAC cell death. To experimentally test this possibility, weestablished stable Bcl-2 and Bcl-xL overexpressing MIA PaCa-2 clones(FIGS. 10A and 10B).

Infection with Ad.5/3-mda-7 did not significantly inhibit cell viabilityin Bcl-2 and Bcl-xL overexpressing MIA PaCa-2 clones, whereassimultaneous treatment with POH resulted in decreased viability, whichinversely correlated with the level of Bcl-2 and Bcl-xL (FIGS. 10C and10D). When Bcl-2 and Bcl-xL overexpressing clones of MIA PaCa-2 wereinfected with Ad.5/3-CTV-M7 and treated with POH, there was asignificant decrease in viability, which was similar in parental andBcl-2 or Bcl-xL overexpressing clones. These results support the utilityof CTV-M7 in treating therapy-resistant PDAC that is mediated byoverexpression of Bcl-2 or Bcl-xL (FIGS. 10C and 10D). Additionally,Bcl-xL overexpressing MIA PaCa-2 clones displayed partial resistancetowards Thapsigargin-induced ER stress-mediated apoptosis as indicatedby decreased PARP cleavage (FIG. 10E). Although Thapsiagargin inducedsimilar levels of BiP/GRP-78 in parental control MIA PaCa-2, modestlyBcl-xL overexpressing MIA Paca-2 cl-2 and high Bcl-xL overexpressing MIAPaCa-2 cl-3, PARP cleavage inversely correlated with the levels ofBcl-xL (FIG. 10E). These results indicate that overexpression of Bcl-xLcan override the increase in BiP/GRP-78 produced by treatment withThapsigargin that leads to extensive PARP cleavage in PDAC cells.Inhibiting BiP/GRP-78 using sh-BiP/GRP-78 reduced apoptosis in MIAPaCa-2 Bcl-xL cl-3 cells (high expresser) treated with Ad.5/3-CTV-M7plus POH (FIG. 10F) without inhibiting production of MDA-7/IL-24 (FIG.10G). Since robust expression of MDA-7/IL-24 by Ad.5/3-CTV in thepresence of POH leads to profound expression of BiP/GRP-78, induction ofpersistent ER stress may result in a switch from pro-survival topro-apoptotic signaling events [57] (FIGS. 10F and 10G). Reducedexpression of BiP/GRP-78 correlated with decreased sensitivity toMDA-7/IL-24-mediated apoptosis (FIGS. 10F and 10G), reinforcing a rolefor sustained ER stress as a mediator of cell death in therapy resistantcells, which could provide an alternative approach to overcome therapyresistance in cancers.

Chimeric Modified CTV-M7 (Ad.5/3-CTV-M7) Eradicates Tumors in MiceBearing Human PDAC Xenografts

A major impediment in cancer gene therapy is the lack of efficientnon-toxic systemic gene delivery systems. Ad and Ad-associated genetherapies have been used efficiently for many years to deliver genes[58] and can be manipulated to replicate specifically in cancer cellswith robust expression of therapeutic genes [7, 11, 29, 38]. A majorobstacle to Ad gene therapy is to provide a means of shielding thetherapeutic Ad (i.e., Ad.mda-7 or CTV-M7) from destruction by the immunesystem and non-specific trapping and clearance in the liver or otherorgan sites not harboring neoplastic cells [28, 59]. To overcome thisproblem, we developed an innovative stealth delivery approach in whichtherapeutic Ads are conjugated with MBs (Ad/MB) and treated withcomplement prior to injection into the tail vein, which shields the Adsin the circulation from trapping in the liver [29] and elimination bythe immune system [28, 30, 59]. To further support the role of MBs in‘stealth delivery’ of Ads, we confirmed that Ads conjugated with MBstreated with complement did not elicit an innate immune response (i.e.,activation of IL-6, TNF-α and IFN-γ) following intravenous tail veininjection into an immune-competent C57B6 mice [30]. In contrast, Adsalone or MB-encapsulated Ads without complement treatment wereimmunogenic [30]. Moreover, complement-treated MBs containing atherapeutic tropism-modified Ad (Ad.5/3-mda-7) coupled with UTMDapproach effectively delivered virus in the tumor region of the prostatein immune-competent transgenic Hi-myc mice resulting in an inhibition intumor development [14].

As a proof-of-principle for site-specific systemic delivery oftherapeutic Ads, nude mice were injected in each flank with 5×10⁶ MIAPaCa-2/luc or MIA PaCa-2/Bcl-xL cells. Tumor-bearing MIA PaCa-2/luc orMIA PaCa-2/Bcl-xL nude mice (n=6 in each group) were then injected intheir tail vein with 100 μl of complement treated Ad/MB (Ad.5/3-vec/MB,Ad.5/3-mda-7/MB, Ad.5/3-PEG-E1A/MB, Ad.5/3-CTV-M7/MB) ˜12 to 14 dayspost cell-implantation. Following tail vein administration of Ad/MB, aportable SonoSite Micro-Maxx US platform equipped with an L25 lineararray transducer set at 0.7 Mechanical Index, 1.8 MPa for 10 min wasused to sonoporate the tumor implanted on the left flank. Mice wereinjected with Ad/MB once a week for 4 wks for a total of fourtreatments, and POH was i.p. administered daily from the first day untilthe 6^(th) week. Some mice were killed 1 day after the last treatmentwith Ad (i.e., 4^(th) week of Ad treatment) to determine the level ofsecreted MDA-7/IL-24 protein in the serum and its potential contributingrole as an anti-tumor agent. Bioluminescene imaging (BLI) was used toquantify tumor size after i.p. administration of 150 mg/kg D-luciferin.There was a positive correlation (r=0.92) of Luminescence intensity vs.cell number (FIG. 16), indicating the utility of BLI in determiningtumor size non-invasively. There was a significant decrease in tumorsizes in both the left and right flanks of mice treated withAd.5/3-CTV-7/MB plus POH as compared to vector infected control(Ad.5/3-vec plus POH) after two weeks of treatment with gene therapy(FIG. 17), and there was no detectable tumor at the end of theexperiment (FIGS. 11A-E and FIG. 17). This may reflect the ability ofMDA-7/IL-24 to promote a ‘bystander’ antitumor effect causing areduction in the growth of the distant tumor not treated with Ad/MB.There was no significant change in dose response curve of MIA PaCa-2/luccells treated with Ad.5-vec+POH as compared to vehicle Ad.5/3-vecindicating that the dose used for POH is a non-toxic but therapeuticallyinactive. Although we found a significant difference in tumor size onthe left sided tumor treated with Ad.5/3-PEG-E1A, no significant changein tumor size was observed in right-flank tumor compared to right-flanktumor of the vehicle-treated group (FIGS. 11B, and 11C).

Using ELISA we observed a higher level of MDA-7/IL-24 in blood plasma ofanimals receiving Ad.5/3-mda-7/MB and Ad.5/3-CTV-M7/MB plus POH ascompared to Ad.5/3-mda-7/MB and Ad.5/3-CTV-M7/MB, respectively, withoutPOH treatment (FIG. 11D). In the context of Ad.5/3-mda-7 plus POH, thiseffect on the distal tumor can be attributed directly to secretedMDA-7/IL-24 and ‘bystander’ antitumor activity in the non-treatedright-sided tumor (FIG. 11E). Since the level of MDA-7/IL-24 protein inthe serum of animals treated with Ad.5/3-CTV-M7/MB plus POH is elevatedin comparison with Ad.5/3-mda-7/MB plus POH, the enhanced effectobserved on the distant tumor may involve ‘bystander’ antitumor activitycaused by secreted MDA-7/IL-24 as well as potential secondary viralinfection of released Ad.5/3-CTV-M7. In contrast, although there wasalmost complete eradication of tumors in the left flank of mice treatedwith Ad.5/3.PEG-E1A alone and with POH, there was a minimal change inthe right-flank tumors (FIGS. 11A, 11B and 11C). This observationfurther validates the superior efficacy of CRCAs armed with atherapeutic cytokine gene (mda-7/IL-24), such as the novelAd.5/3-CTV-M7, that can produce MDA-7/IL-24 which directly kills tumorcells and also affects tumors at distant sites by virtue of ‘bystander’anti-tumors effects, which can eradicate distant PDAC tumors in theopposite flank when animals are treated with POH, i.e., the CGTapproach.

Enhanced Antitumor In Vivo Effect of Chimeric Modified CTV-M7/MB inCombination with POH in Therapy-Resistant PDAC, Potential ClinicalSignificance

Bcl-xL is overexpressed in PDAC and transcriptionally regulated byactivated Ras-Raf-MAPK signaling and as well as NF-κB/STAT3 signalingpathways [60-62] which contributes to chemoresistance [63] and poorprognosis in pancreatic cancer patients [53, 64, 65]. Chemotherapyregimens like 5-flurouracil (5-FU) and gemcitabine are standard firstline therapeutics employed in clinics for the treatment of advancedpancreatic cancers. Repeated exposure of PDAC cells to 5-FU andgemcitabine leads to enhanced anti-apoptotic Bcl-xL expression [63],which eventually can promote chemotherapy resistance in pancreaticcancers, and is a causal factor in disease progression. In order toevaluate a therapy-resistant PDAC model system, we used MIAPaCa-2/Bcl-xL (MIA PaCa-2 cells overexpressing Bcl-xL) to initiatetumors on both flanks of nude mice. Twelve to fourteen days after tumorcell injection, mice were treated as mentioned previously, using MB andultrasound with the UTMD approach, with replication incompetentAd.5/3-mda-7 or replication competent Ad.5/3-CTV-M7 with or without POH.There was a significant decrease in tumor volume as well as tumor massof the sonoporated left-flank in Ad.5/3-CTV-M7 and Ad.5/3-CTV-M7+POHtreated group as compared to Ad.5/3-vec treated group (FIGS. 12A, 12B,12C and 12D). Although we observed a statistically significant decreasein the tumor size of right flank tumors in mice treated withAd.5/3-CTV-M7 alone in the left flank tumors, a profound decrease intumor size of the right flank tumors was observed in those mice treatedwith Ad.5/3-CTV-M7 plus POH. Since this combination induces asignificant increase in the production and secretion of MDA-7/IL-24protein (FIGS. 8, 10A-G and 11A-E), the combinatorial effect of this CGTtreatment supports the prominent role of secreted MDA-7/IL-24 inexecuting its ‘bystander’ effect in reducing growth and killing thesecondary untreated tumor in this therapy-resistant PDAC model (FIG.12A-E). Bcl-xL leads to intrinsic resistance to mda-7/IL-24-mediatedapoptosis in PDAC cells, as observed in prostate cancer cells [39],which was reversed upon treatment with Ad.5/3-CTV-M7. This tumorinhibition was enhanced further in the presence of POH, as shown byTUNEL staining of tumor cells (FIG. 12E).

Discussion

Based on cell culture studies and preclinical animal models, it wasassumed that viruses, particularly viruses that could conditionallyreplicate in cancer cells, would provide ideal weapons to treat cancer[58]. Unfortunately, this promise has not been realized and the majorityof clinical cancer trials using virally administered gene therapies haveproduced only marginal positive results, and responses have not beenenduring. The efficacy of therapeutic viruses has been even moredisappointing in the context of metastatic disease. Newer strategies,including modifying the tropism of viruses to enhance their delivery totumor cells, improved strategies for targeted delivery of therapeuticviruses and development of bipartite viruses that not only replicateselectively in cancer cells but can also produce a therapeutic geneproduct that destroys distant tumor cells through ‘bystander’ activityare bringing us closer to realizing the promise of therapeutic virusesto treat cancer [58]. In order to increase the therapeutic efficacy ofAds by enhancing their infectivity and the ability to deliver transgenesto cancer cells, Ads have been genetically modified resulting inchimeric recombinant Ad.5/3 which displays increased efficacy ininfecting cells irrespective of CAR receptors [2, 13, 31, 66]. Toincrease the therapeutic impact of the Ad.5/3 virus we engineered thisvirus to display cancer-specific replication by using the PEG-Prom [4]and further augmented therapeutic ability by incorporating an additionaltherapeutic gene, either interferon gamma or mda-7/IL-24, referred to asCTVs (CTV-γ and CTV-M7), which would permit distant ‘bystander’anticancer activity [2, 4, 6, 7, 11]. Systemic administration ofAd.5/3-PEG-E1A, a cancer-specific replicating Ad (CRCA), using a MB plusultrasound (UTMD) approach leads to a significant change in tumor volumein a treated tumor, without significantly changing tumor size of asecondary tumor (reflecting a potential metastasis) implanted on theopposite flank, even though these CRCAs theoretically have the potentialto migrate to this distant tumor site in athymic animals. These resultsemphasize the inefficiency of using only a CRCA systemically, suggestingthat the ability to transfer adequate amounts of bioactive CRCAs to adistant secondary tumor site is difficult (and perhaps impossible toachieve, using current vectors and direct systemic delivery approaches).In contrast, further arming of CRCAs with a therapeutic transgene thatis a secreted cytokine, such as mda-7/IL-24 [7, 8, 19], results inimproved clinical responses by the combined effect of oncolysis androbust MDA-7/IL-24 production and secretion [6, 7, 67]. Thus, CTV-M7 canact on distant tumor cells (FIGS. 11A-E and 12A-E) and limits the growthof the distant tumor [2, 7, 67].

Although mda-7/IL-24 effectively induces apoptosis in a wide spectrum ofcancer cells of diverse origin, pancreatic cancer cells are refractoryto mda-7/IL-24-mediated killing due to inhibition of mda-7/IL-24 mRNAtranslation into protein [15, 32, 38]. We previously demonstrated thatROS induced by POH enriched association of mda-7/IL-24 mRNA withpolysomes leading to enhanced translation into MDA-7/IL-24 protein [32].The exact mechanism of action of POH in enhancing the translation ofmda-7/IL-24 mRNA into protein requires further investigation. Earlierpreclinical studies documented chemotherapeutic effects of POH or itsderivatives in inhibiting liver, prostate [68], colon [69] andpancreatic cancer [70], but this effectiveness did not translate inPhase II clinical trials of POH in metastatic prostate [71], metastaticrefractory breast [72] and colon cancer [73]. In most clinical studiesemploying POH, treatment was initiated after the onset of cancer and itsmetastasis, which might be the reason for its ineffective clinicalactivity when used at the prescribed non-toxic dose. It is possible thatPOH could be used to inhibit the initiation of pancreatic cancer(chemoprevention) if taken daily in food supplements in high-risk groups[2], which has not been adequately explored. POH also synergizes withradiotherapy or chemotherapy in inhibiting various cancers [74, 75].Through synergy with other modes of therapy, the clinical dose of POHmight be reduced to a tolerable and achievable physiological level. Weobserved a transient increase in ROS formation even at a lowphysiologically achievable dose of ˜200 μM of POH. This dose issufficient to activate the protein translation machinery by activationof p-70S6K/p4EBP-1 that helps in formation of pre-initiation complex,and thus enhanced MDA-7/IL-24 protein expression from the weaklytranslated mda-7/IL-24 mRNA (unpublished data). This present studyhighlights that using two agents with complementary mechanisms of actionmay prove more efficacious than administering a single agent in thetherapy of pancreatic cancer, and the robust expression of MDA-7/IL-24along with the combined oncolytic effects associated with CTV-M7 plusPOH might be effective in eliminating the residual tumors and thusproviding a way to prevent disease relapse.

Pancreatic cancer is refractory to conventional therapies, which may bea consequence of the accumulation of multiple genetic alterations withdisease progression that is further complicated by the presence ofmetastasis at the time of diagnosis. These genetic mutations often leadto constitutive K-Ras and NF-κB activation, which is associatedwith >90% of PDAC [2, 61]. Ras-Raf-MAPK signaling as well as NF-KB/STAT3signaling pathways lead to transcriptional up-regulation of Bcl-xL inPDAC [60-62], which contributes to chemoresistance [63] and poorprognosis in pancreatic cancer patients [53, 64, 65]. Present resultsindicate that Bcl-xL is significantly downregulated upon mda-7/IL-24expression with maximum decreases when combined with POH treatment,whereas overexpression of Bcl-xL imparts protection tomda-7/IL-24-induced apoptosis in PDAC (FIGS. 8 and 10A-G). Our previousstudies indicated that Bcl-xL differentially protects cancer cells fromMDA-7/IL-24-induced apoptosis [39]. Although Bcl-xL can produceresistance towards MDA-7/IL-24-mediated apoptosis, overexpressingMDA-7/IL-24 by CTVs (which results in higher levels of this cytokine)can circumvent resistance by enhancing and prolonging ER stress thatswitches from pro-survival to pro-apoptotic signaling that leads to celldeath. Our data demonstrates that CRCAs expressing the mda-7/IL-24transgene (Ad.5/3-PEG-E1A-mda-7; Ad.5/3-CTV-M7) reduce PDAC tumors inthe treated left flank of mice as compared to Ad.5/3-vec andAd.5/3-mda-7, which may be due to the oncolytic properties ofAd.5/3-CTV-M7 combined with the apoptosis-promoting properties ofMDA-7/IL-24. Combination CGT therapy with POH and Ad.5/3-CTV-M7 leads toprofound changes in tumor volume in the secondary site (tumors on theopposite flank of animals) as compared to either agent alone in atherapy-resistant in vivo model of PDAC. Apart from the role of POH inpromoting enhanced MDA-7/IL-24 expression, which might initiatesustained ER stress, its role in ROS generation leading to mitochondrialstress may also contribute to enhanced cancer cell killing. Both ERstress and mitochondrial stress might cooperate and promote signaltransduction changes leading to apoptosis even in therapy-resistantPDAC. Through a detailed understanding of precisely how MDA-7/IL-24induces cancer-specific apoptosis, irrespective of genetic diversity intumors, with complementary and additional cellular alterations promotedby POH might provide a viable combinatorial approach in treatingpancreatic cancers where all other therapeutic modalities proveineffectual.

In summary, this Example 2 highlights a chemoprevention gene therapy(CGT) strategy for the effective therapy of PDAC in vitro and in vivo inanimal models with tumors on both flanks. We highlight thatAd.5/3-CTV-M7/MB coupled with the novel UTMD delivery approach [14, 29]as a non-toxic and precise method of targeted gene delivery, which whencombined with POH, which augments the expression of mda-7/IL-24 byfacilitating its translation into protein, sensitizes PDAC tomda-7/IL-24-mediated cytotoxicity, thereby significantly enhancingtherapeutic efficacy. The combinatorial approach is superior toAd.5/3-CTV-M7 alone or POH used as a single modality treatment asphysiologically achievable doses. Based on the safety and diagnosticprofile of MBs, which are now in Phase II and III clinical trials forcardiovascular disease the profound potential clinical value ofAd.5/3-CTV-M7, and the fact that mda-7/IL-24 has been successfullytranslated into the clinic in a Phase I clinical trial [22-26] as safeand efficacious in advanced cancers, this unique CGT strategy may betranslated into the clinic for the treatment of currently incurablepancreatic cancers.

REFERENCES FOR EXAMPLE 2

-   [1] American Cancer Society. Cancer Facts and FIGS. 2012. Atlanta,    Ga.: American Cancer Society Inc. 2012.-   [2] Sarkar S, Azab B M, Das S K, et al. Chemoprevention gene therapy    (CGT): novel combinatorial approach for preventing and treating    pancreatic cancer. Curr Mol Med 2012;-   [3] Garber K. China approves world's first oncolytic virus therapy    for cancer treatment. J Natl Cancer Inst 2006; 98: 298-300.-   [4] Su Z Z, Sarkar D, Emdad L, et al. Targeting gene expression    selectively in cancer cells by using the progression-elevated gene-3    promoter. Proc Natl Acad Sci USA 2005; 102: 1059-64.-   [5] Su Z Z, Shi Y, Fisher P B. Subtraction hybridization identifies    a transformation progression-associated gene PEG-3 with sequence    homology to a growth arrest and DNA damage-inducible gene. Proc Natl    Acad Sci USA 1997; 94: 9125-30.-   [6] Sarkar D, Su Z Z, Park E S, et al. A cancer terminator virus    eradicates both primary and distant human melanomas. Cancer Gene    Ther 2008; 15: 293-302.-   [7] Sarkar D, Su Z Z, Vozhilla N, Park E S, Gupta P, Fisher P B.    Dual cancer-specific targeting strategy cures primary and distant    breast carcinomas in nude mice. Proc Natl Acad Sci USA 2005; 102:    14034-9.-   [8] Sauane M, Gopalkrishnan R V, Sarkar D, et al. MDA-7/IL-24: novel    cancer growth suppressing and apoptosis inducing cytokine. Cytokine    Growth Factor Rev 2003; 14: 35-51.-   [9] Sauane M, Su Z Z, Gupta P, et al. Autocrine regulation of    mda-7/IL-24 mediates cancer-specific apoptosis. Proc Natl Acad Sci    USA 2008; 105: 9763-8.-   [10] Dash R, Bhutia S K, Azab B, et al. mda-7/IL-24: a unique member    of the IL-10 gene family promoting cancer-targeted toxicity.    Cytokine Growth Factor Rev 2010; 21: 381-91.-   [11] Sarkar D, Su Z Z, Vozhilla N, et al. Targeted virus replication    plus immunotherapy eradicates primary and distant pancreatic tumors    in nude mice. Cancer Res 2005; 65: 9056-63.-   [12] Pearson A S, Koch P E, Atkinson N, et al. Factors limiting    adenovirus-mediated gene transfer into human lung and pancreatic    cancer cell lines. Clin Cancer Res 1999; 5: 4208-13.-   [13] Dash R, Dmitriev I, Su Z Z, et al. Enhanced delivery of    mda-7/IL-24 using a serotype chimeric adenovirus (Ad.5/3) improves    therapeutic efficacy in low CAR prostate cancer cells. Cancer Gene    Ther 2010; 17: 447-56.-   [14] Dash R, Azab B, Quinn B A, et al. Apogossypol derivative    BI-97C1 (Sabutoclax) targeting Mcl-1 sensitizes prostate cancer    cells to mda-7/IL-24-mediated toxicity. Proc Natl Acad Sci USA 2011;    108: 8785-90.-   [15] Su Z, Lebedeva I V, Gopalkrishnan R V, et al. A combinatorial    approach for selectively inducing programmed cell death in human    pancreatic cancer cells. Proc Natl Acad Sci USA 2001; 98: 10332-7.-   [16] Lebedeva I V, Emdad L, Su Z Z, et al. mda-7/IL-24, novel    anticancer cytokine: focus on bystander antitumor,    radiosensitization and antiangiogenic properties and overview of the    phase I clinical experience (Review). Int J Oncol 2007; 31:    985-1007.-   [17] Emdad L, Sarkar D, Lebedeva I V, et al. Ionizing radiation    enhances adenoviral vector expressing mda-7/IL-24-mediated apoptosis    in human ovarian cancer. J Cell Physiol 2006; 208: 298-306.-   [18] McKenzie T, Liu Y, Fanale M, Swisher S G, Chada S, Hunt K K.    Combination therapy of Ad-mda7 and trastuzumab increases cell death    in Her-2/neu-overexpressing breast cancer cells. Surgery 2004; 136:    437-42.-   [19] Jiang H, Lin J J, Su Z Z, Goldstein N I, Fisher P B.    Subtraction hybridization identifies a novel melanoma    differentiation associated gene, mda-7, modulated during human    melanoma differentiation, growth and progression. Oncogene 1995; 11:    2477-86.-   [20] Sarkar D, Su Z Z, Lebedeva I V, et al. mda-7 (IL-24) Mediates    selective apoptosis in human melanoma cells by inducing the    coordinated overexpression of the GADD family of genes by means of    p38 MAPK. Proc Natl Acad Sci USA 2002; 99: 10054-9.-   [21] Das S K, Sarkar S, Dash R, et al. Chapter One—Cancer terminator    viruses and approaches for enhancing therapeutic outcomes. Adv    Cancer Res 2012; 115: 1-38.-   [22] Eager R, Harle L, Nemunaitis J. Ad-MDA-7; INGN 241: a review of    preclinical and clinical experience. Expert Opin Biol Ther 2008; 8:    1633-43.-   [23] Tong A W, Nemunaitis J, Su D, et al. Intratumoral injection of    INGN 241, a nonreplicating adenovector expressing the    melanoma-differentiation associated gene-7 (mda-7/IL24): biologic    outcome in advanced cancer patients. Mol Ther 2005; 11: 160-72.-   [24] Fisher P B, Gopalkrishnan R V, Chada S, et al. mda-7/IL-24, a    novel cancer selective apoptosis inducing cytokine gene: from the    laboratory into the clinic. Cancer Biol Ther 2003; 2: S23-37.-   [25] Fisher P B, Sarkar D, Lebedeva I V, et al. Melanoma    differentiation associated gene-7/interleukin-24 (mda-7/IL-24):    novel gene therapeutic for metastatic melanoma. Toxicol Appl    Pharmacol 2007; 224: 300-7.-   [26] Cunningham C C, Chada S, Merritt J A, et al. Clinical and local    biological effects of an intratumoral injection of mda-7 (IL24;    INGN 241) in patients with advanced carcinoma: a phase I study. Mol    Ther 2005; 11: 149-59.-   [27] Hamid O, Varterasian M L, Wadler S, et al. Phase II trial of    intravenous CI-1042 in patients with metastatic colorectal cancer. J    Clin Oncol 2003; 21: 1498-504.-   [28] Muruve D A. The innate immune response to adenovirus vectors.    Hum Gene Ther 2004; 15: 1157-66.-   [29] Greco A, Di Benedetto A, Howard C M, et al. Eradication of    therapy-resistant human prostate tumors using an ultrasound-guided    site-specific cancer terminator virus delivery approach. Mol Ther    2010; 18: 295-306.-   [30] Dash R, Azab B, Shen X N, et al. Developing an effective gene    therapy for prostate cancer: New technologies with potential to    translate from the laboratory into the clinic. Discov Med 2011; 11:    46-56.-   [31] Azab B, Dash R, Das S K, et al. Enhanced delivery of    mda-7/IL-24 using a serotype chimeric adenovirus (Ad.5/3) in    combination with the Apogossypol derivative BI-97C1 (Sabutoclax)    improves therapeutic efficacy in low CAR colorectal cancer cells. J    Cell Physiol 2012; 227: 2145-53.-   [32] Lebedeva I V, Su Z Z, Vozhilla N, et al. Mechanism of in vitro    pancreatic cancer cell growth inhibition by melanoma    differentiation-associated gene-7/interleukin-24 and perillyl    alcohol. Cancer Res 2008; 68: 7439-47.-   [33] Lebedeva I V, Su Z Z, Sarkar D, et al. Induction of reactive    oxygen species renders mutant and wild-type K-ras pancreatic    carcinoma cells susceptible to Ad.mda-7-induced apoptosis. Oncogene    2005; 24: 585-96.-   [34] Bell E L, Klimova T A, Eisenbart J, Schumacker P T, Chandel    N S. Mitochondrial reactive oxygen species trigger hypoxia-inducible    factor-dependent extension of the replicative life span during    hypoxia. Mol Cell Biol 2007; 27: 5737-45.-   [35] Gerasimovskaya E V, Tucker D A, Stenmark K R. Activation of    phosphatidylinositol 3-kinase, Akt, and mammalian target of    rapamycin is necessary for hypoxia-induced pulmonary artery    adventitial fibroblast proliferation. J Appl Physiol 2005; 98:    722-31.-   [36] Huang C, Li J, Ke Q, et al. Ultraviolet-induced phosphorylation    of p70(S6K) at Thr(389) and Thr(421)/Ser(424) involves hydrogen    peroxide and mammalian target of rapamycin but not Akt and atypical    protein kinase C. Cancer Res 2002; 62: 5689-97.-   [37] Bae G U, Seo D W, Kwon H K, et al. Hydrogen peroxide activates    p70(S6k) signaling pathway. J Biol Chem 1999; 274: 32596-602.-   [38] Lebedeva I V, Su Z Z, Vozhilla N, et al. Chemoprevention by    perillyl alcohol coupled with viral gene therapy reduces pancreatic    cancer pathogenesis. Mol Cancer Ther 2008; 7: 2042-50.-   [39] Lebedeva I V, Sarkar D, Su Z Z, et al. Bcl-2 and Bcl-x(L)    differentially protect human prostate cancer cells from induction of    apoptosis by melanoma differentiation associated gene-7,    mda-7/IL-24. Oncogene 2003; 22: 8758-73.-   [40] Webb J L. Effect of more than one inhibitor. Enzyme and    Metabolic Inhibitors. New York: Academic Press; 1963. p. 66-79.-   [41] Chou T C. Theoretical basis, experimental design, and    computerized simulation of synergism and antagonism in drug    combination studies. Pharmacol Rev 2006; 58: 621-81.-   [42] Howard C M, Forsberg F, Minimo C, Liu J B, Merton D A, Claudio    P P. Ultrasound guided site specific gene delivery system using    adenoviral vectors and commercial ultrasound contrast agents. J Cell    Physiol 2006; 209: 413-21.-   [43] Ripple G H, Gould M N, Stewart J A, et al. Phase I clinical    trial of perillyl alcohol administered daily. Clin Cancer Res 1998;    4: 1159-64.-   [44] Hudes G R, Szarka C E, Adams A, et al. Phase I pharmacokinetic    trial of perillyl alcohol (NSC 641066) in patients with refractory    solid malignancies. Clin Cancer Res 2000; 6: 3071-80.-   [45] da Fonseca C O, Simao M, Lins I R, Caetano R O, Futuro D,    Quirico-Santos T. Efficacy of monoterpene perillyl alcohol upon    survival rate of patients with recurrent glioblastoma. J Cancer Res    Clin Oncol 2011; 137: 287-93.-   [46] McGuire K A, Barlan A U, Griffin T M, Wiethoff C M. Adenovirus    type 5 rupture of lysosomes leads to cathepsin B-dependent    mitochondrial stress and production of reactive oxygen species. J    Virol 2011; 85: 10806-13.-   [47] Gupta P, Walter M R, Su Z Z, et al. BiP/GRP78 is an    intracellular target for MDA-7/IL-24 induction of cancer-specific    apoptosis. Cancer Res 2006; 66: 8182-91.-   [48] Wu J, Kaufman R J. From acute ER stress to physiological roles    of the Unfolded Protein Response. Cell Death Differ 2006; 13:    374-84.-   [49] Pataer A, Hu W, Xiaolin L, et al. Adenoviral endoplasmic    reticulum-targeted mda-7/interleukin-24 vector enhances human cancer    cell killing. Mol Cancer Ther 2008; 7: 2528-35.-   [50] Hinz S, Trauzold A, Boenicke L, et al. Bcl-XL protects    pancreatic adenocarcinoma cells against CD95- and    TRAIL-receptor-mediated apoptosis. Oncogene 2000; 19: 5477-86.-   [51] Campani D, Esposito I, Boggi U, et al. Bcl-2 expression in    pancreas development and pancreatic cancer progression. J Pathol    2001; 194: 444-50.-   [52] Evans J D, Cornford P A, Dodson A, Greenhalf W, Foster C S,    Neoptolemos J P. Detailed tissue expression of bcl-2, bax, bak and    bcl-x in the normal human pancreas and in chronic pancreatitis,    ampullary and pancreatic ductal adenocarcinomas. Pancreatology 2001;    1: 254-62.-   [53] Bai J, Sui J, Demirjian A, Vollmer C M, Jr., Marasco W, Callery    M P. Predominant Bcl-XL knockdown disables antiapoptotic mechanisms:    tumor necrosis factor-related apoptosis-inducing ligand-based triple    chemotherapy overcomes chemoresistance in pancreatic cancer cells in    vitro. Cancer Res 2005; 65: 2344-52.-   [54] Carrington E M, McKenzie M D, Jansen E, et al. Islet beta-cells    deficient in Bcl-xL develop but are abnormally sensitive to    apoptotic stimuli. Diabetes 2009; 58: 2316-23.-   [55] Morishima N, Nakanishi K, Tsuchiya K, Shibata T, Seiwa E.    Translocation of Bim to the endoplasmic reticulum (ER) mediates ER    stress signaling for activation of caspase-12 during ER    stress-induced apoptosis. J Biol Chem 2004; 279: 50375-81.-   [56] Zhou Y P, Pena J C, Roe M W, et al. Overexpression of Bcl-x(L)    in beta-cells prevents cell death but impairs mitochondrial signal    for insulin secretion. Am J Physiol Endocrinol Metab 2000; 278:    E340-51.-   [57] Szegezdi E, Logue S E, Gorman A M, Samali A. Mediators of    endoplasmic reticulum stress-induced apoptosis. EMBO Rep 2006; 7:    880-5.-   [58] Curiel D T, Fisher P B. Advances in Cancer Research:    Applications of viruses for cancer therapy. 1st ed 2012. 1-326 p.-   [59] Jiang H, Wang Z, Serra D, Frank M M, Amalfitano A. Recombinant    adenovirus vectors activate the alternative complement pathway,    leading to the binding of human complement protein C3 independent of    anti-ad antibodies. Mol Ther 2004; 10: 1140-2.-   [60] Boucher M J, Morisset J, Vachon P H, Reed J C, Laine J,    Rivard N. MEK/ERK signaling pathway regulates the expression of    Bcl-2, Bcl-X(L), and Mcl-1 and promotes survival of human pancreatic    cancer cells. J Cell Biochem 2000; 79: 355-69.-   [61] Greten F R, Weber C K, Greten T F, et al. Stat3 and NF-kappaB    activation prevents apoptosis in pancreatic carcinogenesis.    Gastroenterology 2002; 123: 2052-63.-   [62] Hamacher R, Schmid R M, Saur D, Schneider G. Apoptotic pathways    in pancreatic ductal adenocarcinoma. Mol Cancer 2008; 7: 64.-   [63] Shi X, Liu S, Kleeff J, Friess H, Buchler M W. Acquired    resistance of pancreatic cancer cells towards 5-Fluorouracil and    gemcitabine is associated with altered expression of    apoptosis-regulating genes. Oncology 2002; 62: 354-62.-   [64] Friess H, Lu Z, Andren-Sandberg A, et al. Moderate activation    of the apoptosis inhibitor bcl-xL worsens the prognosis in    pancreatic cancer. Ann Surg 1998; 228: 780-7.-   [65] Westphal S, Kalthoff H. Apoptosis: targets in pancreatic    cancer. Mol Cancer 2003; 2: 6.-   [66] Hamed H A, Yacoub A, Park M A, et al. Inhibition of multiple    protective signaling pathways and Ad. 5/3 delivery enhances    mda-7/IL-24 therapy of malignant glioma. Mol Ther 2010; 18: 1130-42.-   [67] Chada S, Mhashilkar A M, Ramesh R, et al. Bystander activity of    Ad-mda7: human MDA-7 protein kills melanoma cells via an IL-20    receptor-dependent but STAT3-independent mechanism. Mol Ther 2004;    10: 1085-95.-   [68] Chung B H, Lee H Y, Lee J S, Young C Y. Perillyl alcohol    inhibits the expression and function of the androgen receptor in    human prostate cancer cells. Cancer Lett 2006; 236: 222-8.-   [69] Bardon S, Foussard V, Foumel S, Loubat A. Monoterpenes inhibit    proliferation of human colon cancer cells by modulating cell    cycle-related protein expression. Cancer Lett 2002; 181: 187-94.-   [70] Stark M J, Burke Y D, McKinzie J H, Ayoubi A S, Crowell P L.    Chemotherapy of pancreatic cancer with the monoterpene perillyl    alcohol. Cancer Lett 1995; 96: 15-21.-   [71] Liu G, Oettel K, Bailey H, et al. Phase II trial of perillyl    alcohol (NSC 641066) administered daily in patients with metastatic    androgen independent prostate cancer. Invest New Drugs 2003; 21:    367-72.-   [72] Bailey H H, Attia S, Love R R, et al. Phase II trial of daily    oral perillyl alcohol (NSC 641066) in treatment-refractory    metastatic breast cancer. Cancer Chemother Pharmacol 2008; 62:    149-57.-   [73] Meadows S M, Mulkerin D, Berlin J, et al. Phase II trial of    perillyl alcohol in patients with metastatic colorectal cancer. Int    J Gastrointest Cancer 2002; 32: 125-8.-   [74] Rajesh D, Stenzel R A, Howard S P. Perillyl alcohol as a    radio-/chemosensitizer in malignant glioma. J Biol Chem 2003; 278:    35968-78.-   [75] da Fonseca C O, Linden R, Futuro D, Gattass C R,    Quirico-Santos T. Ras pathway activation in gliomas: a strategic    target for intranasal administration of perillyl alcohol. Arch    Immunol Ther Exp (Warsz) 2008; 56: 267-76.

Example 3. Histone Deacetylase Inhibitors Interact with MDA-7/IL-24 toKill Primary Human Glioblastoma Cells Abstract

We presently demonstrate that histone deacetylase inhibitors (HDACIs)enhance toxicity of melanoma differentiation associatedgene-7/interleukin 24 (mda-7/IL-24) in invasive primary human GBM cells.Additionally, a method is described to augment efficacy of adenoviraldelivery of mda-7/IL-24 in these cells. HDACIs synergized withMDA-7/IL-24 killing GBM cells. Enhanced lethality correlated withincreased autophagy that was dependent on expression of ceramidesynthase 6. HDACIs interacted with MDA-7/IL-24 prolonging generation ofROS and Ca²⁺. Quenching of ROS and Ca²⁺ blocked HDACI and MDA-7/IL-24killing. In vivo MDA-7/IL-24 prolonged survival of animals carryingorthotopic tumors and HDACIs enhanced survival further. A serotype 5/3adenovirus more effectively delivers mda-7/IL-24 to GBM tumors than aserotype 5 virus. Hence, we constructed a serotype 5/3 adenovirus thatconditionally replicates in tumor cells expressing MDA-7/IL-24, in whichthe adenoviral E1A gene was driven by the cancer-specific promoterprogression elevated gene-3 (Ad.5/3-PEG-E1A-mda-7; also calledAd.5/3-CTV; Ad.5/3-CTV-M7). Ad.5/3-CTV (Ad.5/3-CTV-M7) increasedsurvival of mice carrying GBM tumors to a significantly greater extentthan did a non-replicative virus Ad.5/3-mda-7. Ad.5/3-CTV(Ad.5/3-CTV-M7) exhibited no toxicity in the brains of Syrian hamsters.Collectively our data demonstrates that HDACIs enhance MDA-7/IL-24lethality and adenoviral delivery of mda-7/IL-24 combined with tumorspecific viral replication is an effective pre-clinical GBM therapeutic.

Introduction

Glioblastoma multiforme (GBM) is diagnosed in ˜20,000 patients per annum(Robins et al, 2007). Even under circumstances in which virtually all ofthe tumor can be surgically removed and the patients are maximallytreated with radiation and chemotherapy, the mean survival of thisdisease is only extended from 3 months to 1 year (Robins et al, 2007).

The mda-7 gene (Interleukin 24, IL-24) was isolated from human melanomacells induced to undergo differentiation by treatment with interferonand mezerein (Jiang et al, 1995). The expression of MDA-7/IL-24 proteinis decreased in advanced melanomas, with almost undetectable levels inmetastatic disease (Jiang et al, 1995; Ekmekcioglu et al, 2001;Ellerhorst et al, 2002). Enforced expression of MDA-7/IL-24, by use of arecombinant adenovirus Ad.5-mda-7, inhibits the growth and kills a broadspectrum of cancer cells, without exerting harmful effects in normalhuman epithelial or fibroblast cells (Gupta et al, 2006; Lebedeva et al,2005; Fisher et al, 2003; Fisher 2005; Su et al, 2001; Su et al, 1998).Mda-7/IL-24 was evaluated in a Phase I clinical trial in patients withadvanced cancers indicating that an Ad.5-mda-7 (INGN 241) injectedintra-tumorally was safe and with repeated injections, significantclinical activity was evident (Lebedeva et al, 2005; Fisher et al, 2003;Cunningham et al, 2005).

The ability of MDA-7/IL-24 to modulate cell survival processes intransformed cells has been investigated by our groups (Dash et al, 2010;Dent et al, 2010a; Dent et al, 2010b; Bhutia et al, 2011; Sauane et al,2010; Yacoub et al, 2010a; Yacoub et al, 2008a; Yacoub et al, 2008b).Prior work in GBM cells has shown, using bacterially synthesizedGST-MDA-7 protein, that in the low nanomolar concentration rangeGST-MDA-7 primarily causes a growth arrest response with littleinduction of cell killing, whereas at ˜20-fold greater concentrations,the cytokine causes profound growth arrest and tumor cell death (Sauaneet al, 2010; Yacoub et al, 2010a). Key factors implicated in MDA-7/IL-24toxicity included Ca²⁺ elevation, ceramide generation and reactiveoxygen species (ROS) production (Sauane et al, 2010; Yacoub et al,2010). Expression of MDA-7/IL-24 increased the levels of autophagy, andinhibition of autophagy protected against MDA-7/IL-24 toxicity (Yacoubet al, 2010a; Yacoub et al, 2008a; Yacoub et al, 2008b).

Many cancer gene therapy studies have utilized type 5 adenovirus vectors(Curiel and Fisher, 2012). For a type 5 virus to infect a cell requiresexpression of the Coxsackie and Adenovirus receptor (CAR), however, CARis known to be down-regulated in many cancer types including GBM (Curieland Fisher, 2012; Paul et al, 2008). To circumvent the low efficiency oftype 5 adenovirus infection, we created a novel tropism modified vectorby replacing the type 5 virus fiber knob with the fiber knob of the type3 adenovirus resulting in enhanced infection of tumor cells in aCAR-independent manner and our prior pre-clinical studies in prostatecancer and GBM provide evidence for the enhanced therapeutic efficacy ofAd.5/3-mda-7 versus Ad.5-mda-7 (Dash et al, 2010; Hamed et al, 2010).Further studies developed a conditionally replication-competentadenovirus where expression of the adenoviral early region 1A (E1A)virus gene and conditional virus replication was driven by the promoterof progression elevated gene-3 (PEG-3); a promoter which is active onlyin cancer cells, such as GBM cells, but has little activity in normalcells, such as primary astrocytes (Ad.5-PEG-E1A-mda-7; a cancerterminator virus, Ad.5-CTV (Ad.5-CTV-M7) (Su et al, 2005; Sarkar et al,2007; Sarkar et al, 2008). Ad.5-CTV (Ad.5-CTV-M7) injected into prostatecancer or melanoma xenografts in athymic mice eradicated both theprimary infected tumor but also an uninfected tumor growing on theopposite flank (Sarkar et al, 2007; Sarkar et al, 2008). This findingcan be explained by the fact that secreted MDA-7/IL-24 protein,generated from cells infected with Ad.5-mda-7, induces growth inhibitionand apoptosis in surrounding non-infected cancer cells, through a“bystander” anti-tumor effect (Sauane et al, 2008; Emdad et al, 2009).

HDAC inhibitors (HDACIs) are a structurally diverse class of agents,e.g., vorinostat (SAHA; Zolinza) and sodium valproate, (Depakote). Theseagents block histone deacetylation and neutralization of positivelycharged lysine residues on histone tails, thereby modifying chromatinstructure/condensation and transcription (Ellis and Pili, 2010; Spiegelet al, 2012). The mode of HDACI action is in fact multifactorial with anadditional ˜20 targets (Dai et al, 2005; Frew et al, 2009; Tang et al,2012). We have shown that induction of DNA damage and the generation ofceramide and ROS production is a common mechanism involved in bothMDA-7/IL-24 and HDACs-induced anti-tumor activity. As with MDA-7/IL-24,HDACIs have been shown to have selective toxicity in tumor cellscompared to non-transformed cells (Rosato and Grant, 2004).

The present studies were performed to determine whether MDA-7/IL-24 andHDACIs could interact to kill GBM cells and whether a serotype 5/3adenovirus that conditionally replicates in tumor cells expressingMDA-7/IL-24 increased the survival of mice carrying GBM tumors to agreater extent than did a non-replicative virus Ad.5/3-mda-7.

Materials and Methods. Materials.

Suberohydroxamic acid (SBHA) and Vorinostat (SAHA) were supplied byCalbiochem (San Diego, Calif.) as a powder, dissolved in sterile DMSO,and stored frozen under light-protected conditions at −80° C.Trypsin-EDTA, DMEM and RPMI medium, and penicillin-streptomycin werepurchased from GIBCOBRL (GIBCOBRL Life Technologies, Grand Island,N.Y.). Dr. C. D. James, University of California, San Francisco verygenerously originally supplied primary human GBM cells (GBM6, GBM12,GBM14) and information on the genetic background of such cells. Dr. SSpiegel (VCU) supplied the plasmid to express LC3-GFP. Other reagentswere of the highest quality commercially available (Yacoub et al, 2010a;Yacoub et al, 2008a; Yacoub et al, 2008b; Sarkar et al, 2008).

Methods. Generation of Adenoviruses.

Recombinant serotype 5 and serotype 5/3 adenoviruses to expressMDA-7/IL-24 and control empty vector were generated as described inrefs. Dash et al, 2010; Hamed et al, 2010. Ad5/3.PEG-E1.mda-7(Ad.5/3-CTV; Ad.5/3-CTV-M7) was prepared in collaboration with Drs. IgorDmitriev and David Curiel, Washington University School of Medicine inSaint Louis, Mo. This recombinant virus was generated in threeconsecutive steps. 1) Homologous recombination of pAd5/3 genomic plasmidwith pShuttlE3 plasmid containing the mda-7/IL-24 expression cassetteand kanamycin selection resulted in the pAd5/3.E3-mda-7 genome. 2)pAd5/3.E3-mda-7 was cut with Swa I to excise the kanamycin resistancegene. 3) The resultant pAd5/3.E3-mda-7 plasmid was recombined withpShuttlE1 plasmid containing E1A and E1B genes under control of thePEG-3 promoter resulting in Ad5/3.PEG-E1.mda-7 (Ad5/3-CTV;Ad.5/3-CTV-M7) genomic plasmid. This plasmid was digested with Pac I torelease viral ITRs and transfected in A549 cells to rescue the CRCA,Ad.5/3-CTV (Ad.5/3-CTV-M7). Similar strategies were used to generateAd.5/3-cmv-E1A-mda-7 and Ad.5/3-PEG-mda-7. Viruses were expanded andtiters determined as previously described (Sarkar et al, 2005; Azab etal, 2012; Dash et al, 2011).

Cell Culture and In Vitro Exposure of Cells to GST-MDA-7, “Ad.Mda-7” andDrugs.

All GBM lines were cultured at 37° C. (5% (v/v CO₂) in vitro using RPMIsupplemented with 5% (v/v) fetal calf serum and 10% (v/v) Non-essentialamino acids. For short-term cell killing assays and immunoblotting,cells were plated at a density of 3×10³ per cm² and 36 h after platingwere treated with MDA-7/IL-24 and/or various drugs, as indicated. Invitro small molecule inhibitor treatments were from a 100 mM stocksolution of each drug and the maximal concentration of Vehicle (DMSO) inmedia was 0.02% (v/v). For adenoviral infection, cells were infected 12h after plating and the expression of the recombinant viral transgenewas allowed to occur for at least 12 h prior to any additionalexperimental procedure. Cells were not cultured in reduced serum mediaduring any study.

Recombinant Adenoviral Vectors; Infection In Vitro.

We generated and purchased previously noted recombinant serotype 5adenoviruses to express dominant negative caspase 9, c-FLIP-s, CRM A,and BCL-XL (Vector Biolabs, Philadelphia, Pa.). Cells were infected withthese adenoviruses at an approximate m.o.i. of 50. Cells were incubatedfor 24 h to ensure adequate expression of transduced gene products priorto drug exposures.

Detection of Cell Death by Trypan Blue Assays.

Cells were harvested by trypsinization with Trypsin/EDTA for ˜10 min at37° C. As some apoptotic cells detached from the culture substratum intothe medium, these cells were also collected by centrifugation of themedium at 1,500 rpm for 5 min. The pooled cell pellets were resuspendedand mixed with trypan blue dye. Trypan blue stain, in which blue dyeincorporating cells were scored as being dead, was performed by countingof cells using a light microscope and a hemacytometer. Five hundredcells from randomly chosen fields were counted and the number of deadcells was counted and expressed as a percentage of the total number ofcells counted.

Plasmid Transfection.

Plasmid DNA (0.5 μg/total plasmid transfected) was diluted into 50 μl ofRPMI growth media that lacked supplementation with FBS or withpenicillin-streptomycin. Lipofectamine 2000 reagent (1 μl) (Invitrogen,Carlsbad, Calif.) was diluted into 50 μl growth media that lackedsupplementation with FBS or with penicillin-streptomycin. The twosolutions were then mixed together and incubated at room temperature for30 min. The total mix was added to each well (4-well glass slide or12-well plate) containing 200 μl growth media that lackedsupplementation with FBS or with penicillin-streptomycin. The cells wereincubated for 4 h at 37° C., after which time the media was replacedwith RPMI growth media containing 5% (v/v) FBS and 1× pen-strep.

Microscopy for LC3-GFP Expression.

Where indicated LC3-GFP transfected cells, were 12 h after transfectioninfected with either “Ad.cmv” or “Ad.mda-7”, then cultured for 24 h.LC3-GFP transfected cells were visualized at the indicated time pointson the Zeiss Axiovert 200 microscope using the FITC filter.

Intra-Cerebral Inoculation of GBM Cells:

Athymic female NCr-nu/nu mice (NCI-Fredrick) weighing ˜20 g, were usedfor this study. Mice were maintained under pathogen-free conditions infacilities approved by the American Association for Accreditation ofLaboratory Animal Care and in accordance with current regulations andstandards of the U.S. Department of Agriculture, Washington, D.C., theU.S. Department of Health and Human Services, Washington, D.C., and theNational Institutes of Health, Bethesda, Md. GBM cells were cultured inDMEM supplemented with 5% (v/v) fetal calf serum and 100 μg/ml (1% v/v)penicillin-streptomycin. Cells were incubated in a humidified atmosphereof 5% (v/v) CO₂ at 37° C. Mice were anesthetized via i.p. administrationof (ketamine, 40 mg/kg; xylazine, 3 mg/kg) and immobilized in astereotactic frame (KOPF). A 24-gauge needle attached to a Hamiltonsyringe was inserted into the right basal ganglia to a depth of 3.5-mmand then withdrawn 0.5-mm to make space for tumor cell accumulation. Theentry point at the skull was 2-mm lateral and 1-mm dorsal to the bregma.Intra-cerebral injection of 0.5×10⁶ glioma cells (˜40 mice per cell lineper separate experiment) in 2 μl of DMEM medium was performed over 10min. The skull opening was enclosed with sterile bone wax and the skinincision was closed using sterile surgical staples. Adenoviral vectorswere administered seven days after tumor cell implantation viastereotactic injection into the intra-cerebral tumor using the sameanesthesia procedure and stereotactic frame coordinates, as describedabove. Viral vectors suspended in 2 μl of PBS were delivered by slowinfusion over a 6 min period.

Immunohistochemistry and Staining of Fixed Tumor Sections.

Post sacrifice, tumors and associated mouse brains were fixed in OCTcompound (Tissue Tek); cryostat sectioned (Leica) as 12 am sections.Nonspecific binding was blocked with a 2% (v/v) Rat Sera, 1% (v/v)Bovine Sera, 0.1% (v/v) Triton X100, 0.05% (v/v) Tween-20 solution thensections were stained for cell signaling pathway markers: For stainingof sectioned tumors, primary antibodies were applied overnight, sectionswashed with phosphate buffer solution, and secondary antibodies appliedfor detection (as indicated in the Figures): goat anti-rat Alexa 488/647(1:500; Invitrogen); goat anti-mouse Alexa 488/647 (1:500; Invitrogen)secondary antibody as per the primary antibody used as per themanufacturer's instructions. Sections were then de-hydrated, cleared andmounted with cover-slips using Permount. Apoptotic cells with doublestranded DNA breaks were detected using the Upstate TUNEL ApototicDetection Kit according to the manufacturer's instructions. Slides wereapplied to high powered light/confocal microscopes (Zeiss LSM 510Meta-confocal scanning microscope; Zeiss HBO 100 microscope with AxioCam MRm camera) at the indicated magnification in the Figures/Figurelegends. Data shown are representative slides from several sections fromthe same tumor with multiple tumors (from multiple animals; and multipleexperiments) having been examined (n=at least 3-8 animals-tumors).

Data Analysis.

Comparison of the effects of various treatments was performed using oneway analysis of variance and a two tailed Student's t-test. Differenceswith a p-value of <0.05 were considered statistically significant.Statistical examination of in vivo animal survival data utilized logrank statistical analyses between the different treatment groups.Experiments shown are the means of multiple individual points frommultiple experiments (±SEM).

Results

We determined whether HDACIs enhanced MDA-/IL-24 toxicity in primaryhuman GBM cells. GBM6, GBM12 and primary human astrocytes were infectedwith empty vector serotype 5 adenovirus (Ad.5-cmv), or a virus toexpress MDA-7/IL-24 (Ad.5-mda-7). Use of serotype 5 recombinantadenoviruses has been widespread for in vitro use as well as in theclinic. Twelve h after infection cells were treated with increasingdoses of the HDACIs, sodium valproate or suberohydroxamic acid (SBHA).In a dose-dependent fashion treatment with HDACIs enhanced the lethalityof MDA-7/IL-24 in GBM cells but not in primary human astrocytes (FIGS.18A-18C). Drug alone, without infection of control virus, was identicalto that in control virus infected cells (data not shown). In colonyformation assays both valproate and SBHA synergized with MDA-7/IL-24protein to kill GBM cells (Table 1).

TABLE 1 MDA-7/IL-24 synergizes with HDACIs to kill primary human GBMcells. GBM6 cells were plated as single cells (500-2,500 per 60-mm dish,in sextuplicate). Twelve h after plating cells were treated with GST orGST-MDA-7 (10-30 nM), SBHA (1-3 μM) or sodium valproate (0.5-1.5 mM), asindicated. Forty-eight h later cells were washed free of drugs andcolonies were permitted to form for 20 days. (n = 3, +/− SEM). Using theCalcusyn for Windows program we calculated the fraction affected (Fa)and the Combination Index (CI). A CI value of less than 1.00 indicates asynergy of interaction. GST-MDA-7 SBHA GST-MDA-7 Na Val. (nM) (μM) Fa Cl(nM) (mM) Fa Cl 10 1 0.36 0.55 10 0.5 0.29 0.46 20 2 0.60 0.50 20 1.00.40 0.46 30 3 0.81 0.41 30 1.5 0.49 0.37

In a time-dependent fashion MDA-7/IL-24 protein increased autophagy(LC3-GFP vesicle) levels in GBM cells (FIG. 2A). SBHA enhancedMDA-7/IL-24 stimulated autophagy levels; knock down of Beclin1 abolishedautophagy. Knock down of Beclin1 suppressed both MDA-7/IL-24 andMDA-7/IL-24 plus SBHA toxicity (FIG. 19A-B). MDA-7/IL-24 protein andSBHA interacted in a greater than additive fashion to activate PKR-likeendoplasmic reticulum kinase (PERK) and to increase phosphorylation ofthe downstream substrate of PERK, eIF2K (FIG. 19C). Expression ofdominant negative PERK suppressed the induction of autophagy andsuppressed killing by the combination of agents (FIG. 19D and FIG. 20).

Ceramide generation plays a key role in MDA-7/IL-24 lethality, withactivation of the de novo ceramide synthesis pathway (ceramide synthase6 (LASS6)) playing a key role in MDA-7/IL-24-induced ROS levels andchanges in cytosolic Ca²⁺ (Yacoub et al, 2010a). Knock down of LASS6expression suppressed the induction of autophagy in GBM cells andsuppressed killing by the combination of Ad.5-mda-7 and SBHA (FIGS. 21Aand 21B).

We next determined the roles of ROS and changes in cytosolic Ca²⁺ in theresponse of GBM cells to MDA-7/IL-24 and HDACIs. Ad.5-mda-7 and SBHAinteracted in a greater than additive fashion to increase ROS and Ca²⁺levels (FIGS. 22A and 22B). HDACI treatment: (1) increased the initialAd.5-mda-7-induced ROS and Ca²⁺ levels; and (2) prolonged the increasein ROS and Ca²⁺ signaling. Quenching of ROS expressing thioredoxin (TRX)or quenching of Ca²⁺ using calbindin suppressed MDA-7/IL-24 andMDA-7/IL-24 plus SBHA toxicity (FIG. 22C).

GBM cells were infected to express c-FLIP-s and CRM A (inhibitors of theextrinsic apoptosis pathway) or infected to express BCL-XL and dominantnegative caspase 9 (inhibitors of the intrinsic apoptosis pathway).Expression of c-FLIP-s or CRM A did not alter MDA-7/IL-24 toxicity as asingle agent (FIG. 23A). However, expression of c-FLIP-s or CRM Asuppressed the ability of SBHA to enhance MDA-7/IL-24 toxicity.Expression of BCL-XL or dominant negative caspase 9 suppressedMDA-7/IL-24 and MDA-7/IL-24 plus SBHA toxicity. We determined whetherinhibitors of protective BCL-2 family proteins enhanced killing byMDA-7/IL-24. Treatment of GBM cells with either the BCL-2/BCL-XL/MCL-1inhibitor obatoclax or the BCL-2/BCL-XL inhibitor HA14-1 enhanced thelethality of Ad.5-mda-7 (FIG. 23B). These data are similar to priorstudies in prostate cancer cells using the BCL-2 family inhibitorsabutoclax (Azab et al, 2012; Dash et al, 2011).

We developed a tropism modified recombinant adenovirus to expressMDA-7/IL-24 that comprises the tail and shaft domains of a serotype 5adenovirus and the knob domain of a serotype 3 virus (Dash et al, 2010,Hamed et al, 2010). We have published that Ad.5/3-mda-7 prolonged thesurvival of animals carrying GBM tumors to a greater extent than didAd.5-mda-7 (Hamed et al, 2010). GBM cells were implanted into the brainsof athymic mice and tumors infused with virus. In agreement with ourprior publications, infusion of tumors with Ad.5/3-mda-7 prolongedanimal survival (FIG. 24). Treatment of animals with SAHA did notsignificantly enhance animal survival. Combined treatment withAd.5/3-mda-7 and SAHA prolonged survival to a significantly greaterextent than Ad.5.3-mda-7 alone. Collectively our data argue that HDACIsand MDA-7/IL-24 interact to kill GBM cells in vitro and in vivo.

In parallel studies we generated a serotype 5/serotype 3 recombinantadenovirus to express MDA-7/IL-24 that also conditionally replicatesonly in tumor cells (Ad.5/3-PEG-E1A-mda-7; also termed in the Figures asAd.5/3-CTV; Ad.5/3-CTV-M7) (see also Sarkar et al, 2007; Sarkar et al,2008). We compared the growth suppressive effects of Ad.5/3-mda-7 andAd.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV; Ad.5/3-CTV-M7) following infection oforthotopic GBM tumors. GBM6 cells stably transfected to expressluciferase were implanted into athymic nude mouse brains. Seven daysafter implantation mice received a single low dose intra-tumor infusionof recombinant adenovirus. The viruses infused were: Ad.5/3-cmv (emptyvector control, non-replicative); Ad.5/3-PEG-E1A (empty vector control,tumor selective replication); Ad.5/3-mda-7 (MDA-7/IL-24 expression,non-replicative); Ad.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV; Ad.5/3-CTV-M7;MDA-7/IL-24 expression, tumor selective replication). Although a trendwas evident, at the low doses of virus used in this study neitherAd.5/3-PEG-E1A nor Ad.5/3-mda-7 caused a significant decrease in tumorluminosity using a Xenogen IVIS system, i.e., tumor growth (FIG. 25A).However, infusion of Ad.5/3-PEG-E1A-mda-7 (Ad.5/3-CTVK Ad.5/3-CTV-M7)resulted in a significant suppression of tumor mass below the initialvalue.

Animals carrying GBM6-luc tumors were sacrificed, their brains isolated,and immuno-histochemistry performed on the GBM6 tumors within brainsections. Ad.5/3-PEG-E1A caused a modest enhancement in apoptosis/TUNELpositivity in the tumor, an effect which was considerably greater inAd.5/3-mda-7 infected tumors (FIG. 25B). Ad.5/3-PEG-E1A-mda-7(Ad.5/3-CTV-M7)-infected tumors had greater levels of TUNEL positivitythan Ad.5/3-mda-7 infected tumors. In sections of normal brain no TUNELstaining was evident. Infection of tumors with Ad.5/3-PEG-E1A orAd.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV-M7) increased E1A immunoreactivity intumor sections but not in sections of normal brain. This would suggestviral replication and cell killing by Ad. 5/3-PEG-E1A-mda-7(Ad.5/3-CTV-M7) is restricted to tumor tissue.

We next determined using different doses of adenovirus whetherAd.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV-M7) was a more efficacious virus atprolonging animal survival when compared to Ad.5/3-PEG-E1A orAd.5/3-mda-7. At the lowest dose of Ad.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV-M7)tested (1×10⁸ plaque forming units (pfu)), the virus prolonged survivalto a greater extent than did infusion of 1×10⁹ pfu of Ad.5/3-mda-7 (FIG.25C). The intermediate dose of Ad.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV-M7)tested (3×10⁸ pfu) prolonged survival to a greater extent than didinfusion of 1×10⁸ pfu of the same virus. Accordingly, infusion of 1×10⁹pfu of Ad.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV-M7) prolonged survival to agreater extent than did infusion of 3×10⁸ pfu of the same virus, withsome animals living for >250 days. As Ad.5/3-PEG-E1A-mda-7(Ad.5/3-CTV-M7) prolonged animal survival we performed preliminarytoxicology testing of this virus in preparation for its translation intothe clinic. Syrian hamsters are an FDA approved model for oncolyticadenovirus toxicology testing; they are immuno-competent and they permithuman adenovirus replication in normal tissues (Curiel and Fisher, 2012;Dhar et al, 2012). Hamster brains were infused with PBS, an adenovirusthat constitutively replicates Ad.5/3-cmv-E1A (3×10⁹ pfu), orAd.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV-M7) (3×10⁹ pfu). Infusion ofAd.5/3-cmv-E1A strongly increased the levels of TUNEL positivity andexpression of the viral protein E1A in hamster brains (FIG. 25D). Lowlevels of TUNEL positivity and E1A staining were also evident in thelivers of animals who had been infused with Ad.5/3-cmv-E1A; no stainingof the kidneys was observed. In contrast, infection of hamster brainswith Ad.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV-M7) did not increase TUNELpositivity or E1A levels. Staining for MDA-7/IL-24 protein was evidentin Ad.5/3-PEG-E1A-mda-7 infected brains. During our studies we notedthat animals infused with Ad.5/3-cmv-E1A had enlarged neck lymph nodes,indicative that this virus was generating an immune response (FIG. 25E).In contrast, the lymph nodes of animals infused with PBS or withAd.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV-M7) looked identical (and small).

Collectively our in vivo data with Ad.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV-M7)indicate that the virus significantly prolongs animal survival and hasan apparent safe toxicology profile in Syrian hamsters.

Discussion

The research described in this Example 3 has focused on developing noveltherapies for GBM. To achieve these objectives, we utilized mda-7/IL-24,which has demonstrated tumor cell-specific killing andradiosensitization of glioma cells (Yacoub et al, 2010a; Yacoub et al,2008a; Yacoub et al, 2008b; Yacoub et al, 2008c). Prior studies haveshown that inhibition of signaling pathways can enhance MDA-7/IL-24toxicity in GBM cells and the present analyses extended ourcombinatorial approaches targeting histone deacetylases (Yacoub et al,2008b; Hamed et al, 2010). We show that HDACIs increase MDA-7/IL-24toxicity and we also identify a means of enhancing the therapeuticdelivery of MDA-7/IL-24 for GBM using a tropism-modified serotype 5/3adenovirus that conditionally replicates in tumor cells. We are in theadvanced stages of preparation of a Phase I trial dose-limiting toxicitytrial using Ad.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV-M7). In preferredembodiments in GBM, a combination of virotherapy and either HDACinhibitors or, radiotherapy, may be employed.

HDACIs cause oxidative damage to cells which contributes to theirlethality, and possibly to the selectivity of these compounds for tumorcells (Ruefli et al, 2001; Ungerstedt et al, 2005). Our datademonstrated using Ad.5-mda-7 or MDA-7/IL-24 protein in GBM isolatestreated with several HDACIs that the combination of agents synergized tokill GBM cells. Both MDA-7/IL-24 and HDACIs increased the levels of ROSand when combined they further enhanced and prolonged ROS generation.Quenching of ROS suppressed the toxic interaction between the agents. InGBM cells, MDA-7/IL-24 toxicity has been associated with activation ofPERK and the induction of autophagy (Yacoub et al, 2010a, Yacoub et al,2008a; Hamed et al, 2010). HDACIs enhanced both MDA-7/IL-24-inducedactivation of PERK and the increase in autophagy levels. HDACIs assingle agents are known to cause an ER stress response that has beenlinked to acetylation of GRP78/BiP (Kahali et al, 2010): one portion ofthe mechanism by which MDA-7/IL-24 induces ER stress is through bindingto GRP78/BiP (Gupta et al, 2006). Expression of dominant negative PERKor knock down of Beclin1 blocked the increase in autophagy levels andthe toxic interaction between MDA-7/IL-24 and HDACIs. HDACIsdown-regulate c-FLIP-s levels and to increase the levels of deathreceptors (Emanuele et al, 2007). Inhibition of the extrinsic pathwaydid not block MDA-7/IL-24 toxicity however inhibition of this pathwayblunted the interaction between MDA-7/IL-24 and HDACIs. We havepreviously shown in renal and ovarian cancer cells that MDA-7/IL-24lethality is dependent on death receptor signaling (Park et al, 2009;Yacoub et al, 2010b). Inhibition of the intrinsic pathway blocked bothMDA-7/IL-24 and HDACI lethality.

Several HDACIs can cross the blood-brain barrier including sodiumvalproate and vorinostat (Friday et al, 2012). In vivo we noted in micecarrying GBM tumors that Ad.5/3-mda-7 prolonged animal survival and thatthis effect was augmented by HDACI treatment. There have been multiplePhase I and Phase II trials of HDACIs in glioma patients (Friday et al,2012; Lee et al, 2012; Galanis et al, 2009; Chinnaiyan et al, 2012).Alone, although well tolerated by patients vorinostat has modest singleagent activity in GBM, which is in agreement with our findings.Vorinostat has been combined with ionizing radiation, temozolomide,bortezomib and bevacizumab and CPT-11 in GBM patients, with some partialresponses evident (Friday et al, 2012; Lee et al, 2012; Galanis et al,2009; Chinnaiyan et al, 2012).

GBM was one of the earliest malignancies considered amenable to viraldelivery of genetic-based therapeutics (Curiel and Fisher, 2012).Serotype 5 adenoviruses infect through the CAR, a protein whoseexpression is reduced in GBM cells (Curiel and Fisher, 2012; Paul et al,2008; Dash et al, 2010; Hamed et al, 2010). This has resulted in groupsusing targeting strategies to enhance viral infectivity viaCAR-independent pathways. Several laboratories have modified theinfective viral capsid “knob” to bind surface integrin proteins (an RGDmodification) or by insertion into the knob of multiple lysine residues(a pK7 modification) that permit virus attachment to cells through anelectrostatic interactions (Curiel and Fisher, 2012). We have taken theinfective capsid knob from a serotype 3 adenovirus and incorporated itinto the adenovirus type 5 knob; we demonstrated that modified serotype5/3 knob adenoviruses were able to achieve enhanced gene transductioninto low- and high-CAR containing human GBM tumor cells (Curiel andFisher, 2012; Paul et al, 2008; Hamed et al, 2010). We noted that aserotype 5/3 virus was more efficient at transducing genes into GBMcells than either an RGD/double RGD modification or a pK7 modification(Curiel and Fisher, 2012; Hamed et al, 2010).

GBM is a highly invasive and diffuse tumor, which will make infection ofevery tumor cell a difficult and probably an impossible proposition whenusing a non-replicative adenovirus. Many prior studies in GBM have usedserotype 5 viruses, which as mentioned previously, have reducedinfectivity in CAR low GBM cells in situ. In addition to theselimitations, prior gene therapy studies in GBM have also frequentlyexpressed intracellular proteins, e.g. p53, which will result in onlythose cells that have been virally infected being subjected to theactions of the therapeutic gene, i.e., these infections lack a“bystander” secreted protein effect on uninfected tumor cells (Fisher,2005; Dash et al, 2010; Curiel and Fisher, 2012; Hamed et al, 2010;Sauane et al, 2008). In GBM tumors growing on the flanks of mice Ad.5/3-mda-7 therapy suppressed the growth not only of the tumor into whichit was injected but also suppressed growth of the tumor on the oppositeflank (i.e., the un-infected tumor). Thus, consistent with other cancerindications, Ad.5/3-mda-7 generates a “bystander effect” in thecontralateral uninfused GBM tumor (Hamed, Fisher and Dent, unpublishedobservations) (Sauane et al, 2008; Park et al, 2009). Collectively theseconstraints may explain the relative lack of efficacy of previous genetherapy approaches in GBM.

The use of Ad.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV; Ad.5/3-CTV-M7) is oneapproach to overcome the issues of infectivity and the diffuse nature ofGBM. Ad.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV-M7) efficiently infects low CARGBM cells and enhances infectivity even in CAR high GBM cells (Hamed etal, 2010). Ad.5/3-PEG-E1A-mda-7 replicates selectively in tumor cellswhich can result in virus dissemination within the brain to infect tumorcells centimeters away from the site of virus administration.MDA-7/IL-24 protein is secreted from infected GBM cells and astrocytesand as we have recently demonstrated in both GBM, renal and prostatecancer cells media containing secreted MDA-7/IL-24 can induce apoptosisin uninfected tumor cells (Yacoub et al, 2010a; Curiel and Fisher, 2012;Sauane et al, 2008; Park et al, 2009). MDA-7/IL-24 can induce its ownsynthesis in tumor cells, amplifying the initial effect of viralinfection/the initial MDA-7/IL-24 secretion (Curiel and Fisher, 2012;Sauane et al, 2008; Park et al, 2009). Thus, the expression ofMDA-7/IL-24 overcomes the problems associated with a lack of a“bystander” effect following gene therapeutic intervention.

In a dose-dependent fashion Ad.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV-M7)increased animal survival when compared to Ad.5/3-mda-7. At the highestvirus dose tested Ad.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV-M7) prolonged thesurvival of some animals to >250 days. No change in animal behavior orbody mass was noted with these interventions. This data argues thatAd.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV-M7) is a safe and efficacious virus forthe treatment of animal GBM models. Based on these findings, assuggested to us by the FDA, we performed preliminary toxicology testingusing an approved rodent model for human adenovirus replication, theSyrian hamster (Curiel and Fisher, 2012; Dhar et al, 2012). Infusioninto the hamster brain of a constitutively replicating adenovirusAd.5/3-cmv-E1A resulted in significant levels of apoptosis andexpression of the viral protein E1A; thus viral replication hadoccurred. The liver is a major site of adenovirus clearance from theblood, and following infusion of this virus into the brain we noted lowlevels of apoptosis and E1A expression in the liver. The kidneys did notexhibit any virus uptake. Infusion into the brain ofAd.5/3-PEG-E1A-mda-7 (Ad.5/3-CTV-M7) did not result in apoptosis orexpression of the viral E1A protein. This argues that control of virusreplication by the PEG-3 promoter was tumor cell specific. Syrianhamsters are an immune-competent model and would be expected toimmunologically respond to virus replication (Curiel and Fisher, 2012;Dhar et al, 2012). In agreement with this hypothesis we found that necklymph nodes were enlarged in Ad.5/3-cmv-E1A infected animals, whereasinfusion of Ad.5/3-PEG-E1A-mda-7 had no obvious effect on lymph nodesize. These findings further demonstrate that Ad.5/3-PEG-E1A-mda-7(Ad.5/3-CTV-M7) is a safe and efficacious virus in vivo.

Our data demonstrate that HDACIs increase MDA-7/IL-24 lethality throughmechanisms involving ER stress and activation of the extrinsic apoptosispathway. Adenoviral delivery of mda-7/IL-24 to GBM cells and tumors canbe enhanced by a serotype 3 tropism modification and by engineering ofthe virus to conditionally replicate in tumor cells.

REFERENCES FOR EXAMPLE 3

-   Azab B, Dash R, Das S K, Bhutia S K, Shen X N, Quinn B A, et    al. (2012) Enhanced delivery of mda-7/IL-24 using a serotype    chimeric adenovirus (Ad.5/3) in combination with the Apogossypol    derivative BI-97C1 (Sabutoclax) improves therapeutic efficacy in low    CAR colorectal cancer cells. J Cell Physiol. 227: 2145-2153.-   Bhutia S K, Das S K, Azab B, Dash R, Su Z Z, Lee S G, et al. (2011)    Autophagy switches to apoptosis in prostate cancer cells infected    with melanoma differentiation associated gene-7/interleukin-24    (mda-7/IL-24). Autophagy. 7: 1076-1077.-   Chinnaiyan P, Chowdhary S, Potthast L, Prabhu A, Tsai Y Y, Sarcar B,    et al. (2012) Phase I trial of vorinostat combined with bevacizumab    and CPT-11 in recurrent glioblastoma. Neuro Oncol. 14: 93-100.-   Cunningham C C, Chada S, Merritt J A, Tong A, Senzer N, Zhang Y, et    al. (2005) Clinical and local biological effects of an intratumoral    injection of mda-7 (IL24; INGN 241) in patients with advanced    carcinoma: a phase I study. Mol Ther 11: 149-159.-   Curiel D T, Fisher P B. (Eds.) (2012) Applications of Viruses for    Cancer Therapy. Adv Cancer Res. 115: 1-334.-   Dai Y, Rahmani M, Dent P, Grant S. (2005) Blockade of histone    deacetylase inhibitor-induced RelA/p65 acetylation and NF-kappaB    activation potentiates apoptosis in leukemia cells through a process    mediated by oxidative damage, XIAP downregulation, and c-Jun    N-terminal kinase 1 activation. Mol Cell Biol. 25: 5429-5444.-   Dash R, Bhutia S K, Azab B, Su Z Z, Quinn B A, Kegelmen T P, et    al. (2010) mda-7/IL-24: a unique member of the IL-10 gene family    promoting cancer-targeted toxicity. Cytokine Growth Factor Rev. 21:    381-391.-   Dash R, Azab B, Quinn B A, Shen X, Wang X Y, Das S K, et al. (2011)    Apogossypol derivative BI-97C1 (Sabutoclax) targeting Mcl-1    sensitizes prostate cancer cells to mda-7/IL-24-mediated toxicity.    Proc Natl Acad Sci USA. 108: 8785-8790.-   Dash R, Dmitriev I, Su Z Z, Bhutia S K, Azab B, Vozhilla N, et    al. (2010) Enhanced delivery of mda-7/IL-24 using a serotype    chimeric adenovirus (Ad.5/3) improves therapeutic efficacy in low    CAR prostate cancer cells. Cancer Gene Ther. 17: 447-456.-   Dhar D, Toth K, Wold W S. (2012) Syrian hamster tumor model to study    oncolytic Ad5-based vectors. Methods Mol Biol. 797:53-63.-   Dent P, Yacoub A, Hamed H A, Park M A, Dash R, Bhutia S K, et al.    (2010a) The development of MDA-7/IL-24 as a cancer therapeutic.    Pharmacol Ther. 128: 375-384.-   Dent P, Yacoub A, Hamed H A, Park M A, Dash R, Bhutia S K, et al.    (2010b) MDA-7/IL-24 as a cancer therapeutic: from bench to bedside.    Anticancer Drugs. 21: 725-731.-   Ekmekcioglu S, Ellerhorst J, Mhashilkar A M, Sahin A A, Read C M,    Prieto V G, et al. (2001) Down-regulated melanoma differentiation    associated gene (mda-7) expression in human melanomas. Int J Cancer    94: 54-59.-   Ellerhorst J A, Prieto V G, Ekmekcioglu S, Broemeling L, Yekell S,    Chada S, et al. (2002) Loss of MDA-7 expression with progression of    melanoma. J Clin Oncol 20: 1069-1074.-   Ellis L, Pili R. (2010) Histone Deacetylase Inhibitors: Advancing    Therapeutic Strategies in Hematological and Solid Malignancies.    Pharmaceuticals (Basel) 3: 2411-69.-   Emanuele S, Lauricella M, Carlisi D, Vassallo B, D'Anneo A, Di Fazio    P, et al. (2007) SAHA induces apoptosis in hepatoma cells and    synergistically interacts with the proteasome inhibitor Bortezomib.    Apoptosis. 12: 1327-1338.-   Emdad L, Lebedeva I V, Su Z Z, Gupta P, Sauane M, Dash R, et    al. (2009) Historical perspective and recent insights into our    understanding of the molecular and biochemical basis of the    antitumor properties of mda-7/IL-24. Cancer Biol Ther. 8: 391-400.-   Fisher P B, Gopalkrishnan R V, Chada S, Chada S, Ramesh R, Grimm E    A, et al. (2003) mda-7/IL-24, a novel cancer selective apoptosis    inducing cytokine gene: from the laboratory into the clinic. Cancer    Biol Ther 2: S23-37.-   Fisher P B. (2005) Is mda-7/IL-24 a “magic bullet” for cancer?    Cancer Res 65: 10128-10138.-   Frew A J, Johnstone R W, Bolden J E. (2009) Enhancing the apoptotic    and therapeutic effects of HDAC inhibitors. Cancer Lett 280: 125-33.-   Friday B B, Anderson S K, Buckner J, Yu C, Giannini C, Geoffroy F,    et al. (2012) Phase II trial of vorinostat in combination with    bortezomib in recurrent glioblastoma: a north central cancer    treatment group study. Neuro Oncol. 14:215-221.-   Galanis E, Jaeckle K A, Maurer M J, Reid J M, Ames M M, Hardwick J    S, et al. (2009) Phase II trial of vorinostat in recurrent    glioblastoma multiforme: a north central cancer treatment group    study. J Clin Oncol. 27: 2052-2058.-   Gupta P, Su Z Z, Lebedeva I V, Sarkar D, Sauane M, Emdad L, et    al. (2006) mda-7/IL-24: multifunctional cancer-specific    apoptosis-inducing cytokine. Pharmacol Ther 111: 596-628.-   Gupta P, Walter M R, Su Z Z, Lebedeva I V, Emdad L, Randolph A, et    al. (2006) BiP/GRP78 is an intracellular target for MDA-7/IL-24    induction of cancer-specific apoptosis. Cancer Res. 66: 8182-8191.-   Hamed H A, Yacoub A, Park M A, Eulitt P J, Dash R, Sarkar D, et    al. (2010) Inhibition of multiple protective signaling pathways and    Ad.5/3 delivery enhances mda-7/IL-24 therapy of malignant glioma.    Mol Ther. 18: 1130-1142.-   Jiang H, Lin J J, Su Z Z, Goldstein, N I, Fisher, P B. (1995)    Subtraction hybridization identifies a novel melanoma    differentiation associated gene, mda-7, modulated during human    melanoma differentiation, growth and progression. Oncogene 11:    2477-2486.-   Kahali S, Sarcar B, Fang B, Williams E S, Koomen J M, Tofilon P J,    et al. (2010) Activation of the unfolded protein response    contributes toward the antitumor activity of vorinostat. Neoplasia.    12: 80-86.-   Lebedeva I V, Sauane M, Gopalkrishnan R V, Sarkar D, Su Z Z, Gupta    P, et al. (2005) mda-7/IL-24: exploiting cancer's Achilles' heel.    Mol Ther 11: 4-18.-   Lee E Q, Puduvalli V K, Reid J M, Kuhn J G, Lamborn K R, Cloughesy T    F, et al. (2012) Phase I study of vorinostat in combination with    temozolomide in patients with high-grade gliomas: North American    Brain Tumor Consortium Study 04-03. Clin Cancer Res. 18: 6032-6039.-   Park M A, Walker T, Martin A P, Allegood J, Vozhilla N, Emdad L, et    al. (2009) MDA-7/IL-24-induced cell killing in malignant renal    carcinoma cells occurs by a ceramide/CD95/PERK-dependent mechanism.    Mol Cancer Ther. 8: 1280-1291.-   Paul C P, Everts M, Glasgow J N, Dent P, Fisher P B, Ulasov I V, et    al. (2008) Characterization of infectivity of knob-modified    adenoviral vectors in glioma. Cancer Biol Ther. 7: 786-793.-   Robins H I, Chang S, Butowski N, Mehta M. (2007) Therapeutic    advances for glioblastoma multiforme: current status and future    prospects. Curr Oncol Rep 9: 66-70.-   Rosato R R and Grant S. (2004) Histone deacetylase inhibitors in    clinical development. Expert Opin Investig Drugs 13: 21-38.-   Ruefli A A, Ausserlechner M J, Bernhard D, Sutton V R, Tainton K M,    Kofler R, et al. (2001) The histone deacetylase inhibitor and    chemotherapeutic agent suberoylanilide hydroxamic acid (SAHA)    induces a cell-death pathway characterized by cleavage of Bid and    production of reactive oxygen species. Proc Natl Acad Sci USA. 98:    10833-10838.-   Sarkar D, Lebedeva I V, Su Z Z, Park E S, Chatman L, Vozhilla N, et    al. (2007) Eradication of therapy-resistant human prostate tumors    using a cancer terminator virus. Cancer Res. 67: 5434-5442.-   Sarkar D, Su Z Z, Park E S, Vozhilla N, Dent P, Curiel D T, et    al. (2008) A cancer terminator virus eradicates both primary and    distant human melanomas. Cancer Gene Ther. 15: 293-302.-   Sarkar D, Su Z-z, Vozhilla N, Park E S, Gupta P, Fisher P B. (2005)    Dual cancer-specific targeting strategy cures primary and distant    breast carcinomas in nude mice. Proc Natl Acad Sci USA. 102:    14034-14039.-   Sauane M, Su Z Z, Dash R, Liu X, Norris J S, Sarkar D, et al. (2010)    Ceramide plays a prominent role in MDA-7/IL-24-induced    cancer-specific apoptosis. J Cell Physiol. 222: 546-555.-   Sauane M, Su Z Z, Gupta P, Lebedeva I V, Dent P, Sarkar D, et    al. (2008) Autocrine regulation of mda-7/IL-24 mediates    cancer-specific apoptosis. Proc Natl Acad Sci USA. 105: 9763-9768.-   Spiegel S, Milstien S, Grant S. (2012) Endogenous modulators and    pharmacological inhibitors of histone deacetylases in cancer    therapy. Oncogene. 31: 537-551.-   Su Z, Lebedeva I V, Gopalkrishnan R V, Goldstein N I, Stein C A,    Reed J C, et al. (2001) A combinatorial approach for selectively    inducing programmed cell death in human pancreatic cancer cells.    Proc Natl Acad Sci USA 98: 10332-10337.-   Su Z Z, Madireddi M T, Lin J J, Young C S, Kitada S, Reed J C, et    al. (1998) The cancer growth suppressor gene mda-7 selectively    induces apoptosis in human breast cancer cells and inhibits tumor    growth in nude mice. Proc Natl Acad Sci USA 95: 14400-14405.-   Su Z Z, Sarkar D, Emdad L, Duigou G J, Young C S, Ware J, et    al. (2005) Targeting gene expression selectively in cancer cells by    using the progression-elevated gene-3 promoter. Proc Natl Acad Sci    USA. 102: 1059-1064.-   Tang Y, Yacoub A, Hamed H A, Poklepovic A, Tye G, Grant S, et    al. (2012) Sorafenib and HDAC inhibitors synergize to kill CNS tumor    cells. Cancer Biol Ther. 13: 567-574.-   Ungerstedt J S, Sowa Y, Xu W S, Shao Y, Dokmanovic M, Perez G, et    al. (2005) Role of thioredoxin in the response of normal and    transformed cells to histone deacetylase inhibitors. Proc Natl Acad    Sci USA. 102: 673-678.-   Yacoub A, Hamed H A, Allegood J, Mitchell C, Spiegel S, Lesniak M S,    et al. (2010a) PERK-dependent regulation of ceramide synthase 6 and    thioredoxin play a key role in mda-7/IL-24-induced killing of    primary human glioblastoma multiforme cells. Cancer Res. 70:    1120-1129.-   Yacoub A, Park M A, Gupta P, Rahmani M, Zhang G, Hamed H, et al.    (2008a) Caspase, cathepsin-, and PERK-dependent regulation of    MDA-7/IL-24-induced cell killing in primary human glioma cells. Mol    Cancer Ther. 7: 297-313.-   Yacoub A, Gupta P, Park M A, Rhamani M, Hamed H, Hanna D, et al.    (2008b) Regulation of GST-MDA-7 toxicity in human glioblastoma cells    by ERBB1, ERK1/2, PI3K, and JNK1-3 pathway signaling. Mol Cancer    Ther. 7: 314-329.-   Yacoub A, Hamed H, Emdad L, Dos Santos W, Gupta P, Broaddus W C, et    al. (2008c) MDA-7/IL-24 plus radiation enhance survival in animals    with intracranial primary human GBM tumors. Cancer Biol Ther. 7:    917-933.-   Yacoub A, Liu R, Park M A, Hamed H A, Dash R, Schramm D N, et al.    (2010b) Cisplatin enhances protein kinase R-like endoplasmic    reticulum kinase- and CD95-dependent melanoma    differentiation-associated gene-7/interleukin-24-induced killing in    ovarian carcinoma cells. Mol Pharmacol. 77: 298-310.

While the invention has been described in terms of its preferredembodiments, those skilled in the art will recognize that the inventioncan be practiced with modification within the spirit and scope of theappended claims. Accordingly, the present invention should not belimited to the embodiments as described above, but should furtherinclude all modifications and equivalents thereof within the spirit andscope of the description provided herein.

1.-17. (canceled)
 18. A method of treating cancer in a subject in needthereof, the method comprising administering to the subject atherapeutically effective amount of a composition comprising anadenovirus, wherein: (a) the adenovirus comprises a genome encoding amelanoma differentiation associated gene-7/interleukin-24 (mda-7/IL-24)protein; (b) the adenovirus binds desmoglein-2 or CD46; (c) theadenovirus is replication competent in cancer cells; and (d) the canceris a prostate cancer, a pancreatic cancer, or a glioblastoma.
 19. Themethod of claim 18, wherein the mda-7/IL-24 protein is encoded within anE3 region of the adenovirus genome.
 20. The method of claim 18, whereinthe adenovirus comprises a viral capsid knob of a serotype 3 adenovirus.21. The method of claim 18, wherein the adenovirus is a recombinantserotype 5 adenovirus.
 22. The method of claim 21, wherein theadenovirus is an adenovirus serotype 5/serotype 3 chimera (Ad.5/3). 23.The method of claim 18, wherein viral replication of the adenovirus isunder the control of a cancer-selective promoter.
 24. The method ofclaim 23, wherein the cancer-selective promoter is a ProgressionElevated Gene (PEG)-3 promoter.
 25. The method of claim 18, wherein saidadministering is performed systemically.
 26. The method of claim 25,wherein said virus is encapsulated in microbubbles in a physiologicallyacceptable carrier.
 27. The method of claim 18, wherein the cancer is aprostate cancer.
 28. The method of claim 18, wherein the cancer is apancreatic cancer.
 29. The method of claim 18, wherein the cancer is aglioblastoma.
 30. The method of claim 18, further comprisingadministering to the patient at least one additional therapeutic agent.31. The method of claim 30, wherein said at least one additionaltherapeutic agent is selected from the group consisting of an agent thataugments reactive oxygen (ROS) production, an HDAC inhibitor, and anMCL-1 inhibitor.
 32. The method of claim 30, wherein the additionalagent is perillyl alcohol.
 33. The method of claim 32, wherein thecancer is a pancreatic cancer.
 34. The method of claim 30, wherein theadditional agent is sodium valproate or suberohydroxamic acid.
 35. Themethod of claim 34, wherein the cancer is a glioblastoma.
 36. The methodof claim 30, wherein the additional agent is sabutoclax.
 37. The methodof claim 36, wherein the cancer is a prostate cancer.